Evolution of the newest diagnostic methods for COVID-19: a Chinese perspective 中国视角下的新冠肺炎最新诊断方法演进 1 equal first-author 2 equal 1 1 3 3 3 2 corresp equal Huimin HUANG, huimin@gird.cn Huimin HUANG, huimin@gird.cn huimin@gird.cn https://orcid.org/0000-0002-7948-3011 1 corresp equal Baoqing SUN, sunbaoqing@vip.163.com Baoqing SUN, sunbaoqing@vip.163.com sunbaoqing@vip.163.com https://orcid.org/0000-0002-1671-0723 广州医科大学附属第一医院， 广州呼吸健康研究院， 呼吸疾病国家重点实验室， 国家呼吸疾病临床医学中心， 国家呼吸医学中心， 检验科， 中国广州市， 510140 广州医科大学附属第一医院， 广州呼吸健康研究院， 呼吸疾病国家重点实验室， 国家呼吸疾病临床医学中心， 国家呼吸医学中心， 检验科， 中国广州市， 510140 Department of Clinical Laboratory, National Center for Respiratory Medicine, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510140, China Department of Clinical Laboratory, National Center for Respiratory Medicine, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510140, China 广州医科大学附属第一医院， 广州呼吸健康研究院， 呼吸疾病国家重点实验室， 国家呼吸疾病临床医学中心， 国家呼吸医学中心， 中国广州市， 510140 广州医科大学附属第一医院， 广州呼吸健康研究院， 呼吸疾病国家重点实验室， 国家呼吸疾病临床医学中心， 国家呼吸医学中心， 中国广州市， 510140 National Center for Respiratory Medicine, National Clinical Research Center of Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510140, China National Center for Respiratory Medicine, National Clinical Research Center of Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510140, China 广州医科大学， 中国广州市， 511495 广州医科大学， 中国广州市， 511495 Guangzhou Medical University, Guangzhou 511495, China Guangzhou Medical University, Guangzhou 511495, China Coronavirus disease 2019 (COVID-19) has continued to spread globally since late 2019, representing a formidable challenge to the world’s healthcare systems, wreaking havoc, and spreading rapidly through human contact. With fever, fatigue, and a persistent dry cough being the hallmark symptoms, this disease threatened to destabilize the delicate balance of our global community. Rapid and accurate diagnosis of COVID-19 is a prerequisite for understanding the number of confirmed cases in the world or a region, and an important factor in epidemic assessment and the development of control measures. It also plays a crucial role in ensuring that patients receive the appropriate medical treatment, leading to optimal patient care. Reverse transcription-polymerase chain reaction (RT-PCR) technology is currently the most mature method for detecting viral nucleic acids, but it has many drawbacks. Meanwhile, a variety of COVID-19 detection methods, including molecular biological diagnostic, immunodiagnostic, imaging, and artificial intelligence methods have been developed and applied in clinical practice to meet diverse scenarios and needs. These methods can help clinicians diagnose and treat COVID-19 patients. This review describes the variety of such methods used in China, providing an important reference in the field of the clinical diagnosis of COVID-19. 自2019年底以来，新型冠状病毒感染（COVID-19）继续在全球蔓延，对世界卫生保健系统构成严峻挑战，造成严重破坏，并通过人类接触迅速传播。这种疾病的主要症状是发烧、疲劳和持续的干咳，它威胁到我们全球社会健康系统的平衡。快速准确诊断新冠肺炎是掌握全球或地区确诊病例数量的前提，也是疫情评估和制定控制措施的重要因素。同时，这也确保患者获得合理治疗，为患者获得最佳的护理方案发挥着至关重要的作用。逆转录聚合酶链反应（RT-PCR）技术是目前检测病毒核酸最成熟的方法，但它存在很多缺点。与此同时，分子生物学诊断、免疫诊断、影像学、人工智能等多种新型冠状病毒检测方法已经开发并应用于临床，适应不同场景和需求。这些方法可以帮助临床医生诊断和治疗COVID-19患者。本文总结了目前国内常用于新冠肺炎诊断的多种最新检测方法，为新冠肺炎临床诊断提供了重要参考。 SARS-CoV-2 COVID-19 诊断 聚合酶链式反应（PCR） 免疫分析 影像学 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Coronavirus disease 2019 (COVID-19) Diagnosis Polymerase chain reaction (PCR) Immunoassay Radiography 1 Introduction 1 The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a significant threat to global public health systems ( Cheng and Shan, 2020 Cheng and Shan, 2020 ). SARS-CoV-2 has wreaked havoc on global balance due to its worldwide transmission primarily through person-to-person contact, with fever, fatigue, and a dry cough being the most common symptoms ( Wen et al., 2020 Wen et al., 2020 ; Bai and Tao, 2021 Bai and Tao, 2021 ; Islam et al., 2023c Islam et al., 2023c ). According to Our World in Data ( https://ourworldindata. https://ourworldindata. ‍org/explorers/coronavirus-data-explorer), as of February 20, 2023, more than 686 million people have been affected and 6.74 million people have died from coronavirus disease 2019 (COVID-19) infections. Since the outbreak of COVID-19, the SARS-CoV-2 virus has mutated and various variants such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.6, B.1.6.2), and Omicron (B.1.1.529) have been identified as variants of interest and concern ( Sakib et al., 2021 Sakib et al., 2021 ; Islam et al., 2022d Islam et al., 2022d , 2023c 2023c ; Soto et al., 2023 Soto et al., 2023 ). Recently, XBB.1.5, a subvariant of the recombinant mutant XBB, has rapidly become the dominant SARS-CoV-2 strain in the USA and has now been detected in mainland China ( Yue et al., 2023 Yue et al., 2023 ). Like XBB.1, XBB.1.5 can evade neutralization by plasma and serum from vaccinated or convalescent individuals and monoclonal antibodies (mAbs) ( Cao et al., 2023 Cao et al., 2023 ; Kurhade et al., 2023 Kurhade et al., 2023 ). However, some patients have not exhibited obvious clinical symptoms of infection in the early stages. These asymptomatic infections account for about 40% to 45% of SARS-CoV-2 infections, and have an even stronger and more persistent ability to transmit the virus ( Al-Tawfiq, 2020 Al-Tawfiq, 2020 ; Oran and Topol, 2020 Oran and Topol, 2020 ), so early detection, isolation, and treatment are critical for reducing morbidity and mortality. This ongoing global pandemic is causing researchers to consider new approaches to detect, diagnose, and treat COVID-19. Recently, wastewater-based epidemiological monitoring (WBEM) has become an efficient and feasible surveillance protocol in a pandemic such as COVID-19. WBEM can be combined with clinical samples for use in early warning of outbreaks, tracking the current trend, detection of genetic diversity and symptomatic individuals, deploying mass vaccination including booster doses, as well as detecting an up-surge in new SARS-CoV-2 variants ( Jakariya et al., 2022 Jakariya et al., 2022 ; Islam et al., 2023a Islam et al., 2023a , 2023c 2023c ). The salient findings of such studies indicate that monitoring the genetic markers of SARS-CoV-2 in wastewater can identify COVID-19 cases, which reduces the burden on the public health system during pandemics. In addition, during the COVID-19 pandemic, monkeypox virus (MPXV) and Langya virus (LayV) are other zoonoses have re-emerged, which can spread dramatically either between humans or from animals to humans by bite/scratch, close contact, or by eating undercooked meat from infected animals. These re-emerging viral diseases have spread swiftly in several countries and constitute an ongoing global public health emergency. Many studies have shown that polymerase chain reaction (PCR) using a specific primer could be a gold standard method for diagnosis of zoonotic diseases, while other diagnostic methods should be investigated further ( Chakraborty et al., 2022 Chakraborty et al., 2022 ; Chandran et al., 2022 Chandran et al., 2022 ; Islam et al., 2022c Islam et al., 2022c , 2023b 2023b ). To detect SARS-CoV-2 quickly and accurately, the current mainstream method is real-time reverse transcription-PCR (RT-PCR) ( Huang et al., 2020 Huang et al., 2020 ). However, because the test is influenced by the sample materials, handling method, and transport, the sensitivity of the test results fluctuates. There is still an urgent need to improve the sensitivity and specificity of detection, so more accurate and diversified genetic tests are emerging. Serological detection is a good supplement to alleviate the deficiencies of PCR for monitoring the level of antibodies after infection and vaccination. Meanwhile, we should try alternatives to clinical tests for COVID-19, such as combining WBEM and clinical trials further. Imaging examination can also provide the basis for clinical condition monitoring. The goal of this study was to introduce various detection and diagnostic methods at the molecular, immunological, and digital levels, and analyze their utilization in the detection and diagnosis of COVID-19. We focus on the evolution in diagnostic approaches in China, from the beginning of the outbreak to the current stage. We also aimed to better understand the feasibility and acceptability of a variety of diagnostic methods during the pandemic, and address how this understanding has aided the development of more suitable applications for different scenarios. Fig. 1 Fig. 1 shows schematics of common detection techniques that will be discussed in detail ( Islam et al., 2022a Islam et al., 2022a , 2022b 2022b , 2022e 2022e ). 2 Clinical manifestation 1 Fever, chills, cough, shortness of breath, dyspnea, expectoration, headache, nausea, vomiting, muscle pain, joint pain, weakness, fatigue, and other symptoms are common in COVID-19 patients ( Yang et al., 2020 Yang et al., 2020 ). Fever, cough, and fatigue are the three most common in patients, with fever being the most serious. About 90% of patients exhibit more than one clinical symptom. Following COVID-19 infection, patients show hematologic signs of leukopenia, lymphocytopenia, and decreased platelet counts, as well as decreased cluster of differentiation 4-positive (CD4 + ) and CD8 + T lymphocyte subsets in peripheral blood, eosinophil reduction in most patients, decreased hemoglobin solubility to blood cells, and abnormal coagulation indicators ( Shen et al., 2020 Shen et al., 2020 ). In addition to cough and fever caused by lung inflammation, COVID-19 patients exhibit clinical symptoms in the gastrointestinal tract, liver, nerves, kidneys, and eyes. The most common and critical febrile symptoms of COVID-19 patients can be used as crude screening criteria in the general population. Furthermore, the sudden loss of smell and taste has been linked to the possibility of COVID-19 infection, providing a smoking gun for COVID-19. Early symptoms, such as eye symptoms, coughing, and fatigue, may provide a preliminary clinical diagnosis. Patients with certain organ or system injuries, such as gastrointestinal tract and eye injuries, may have a more severe course of the disease, which could guide treatment methods to a certain extent. Hematological manifestations such as lymphocytopenia, leucopenia, and thrombocytopenia may aid clinical detection. Brodin (2021) Brodin (2021) discovered that children under the age of 18 are less likely to encounter severe symptoms of COVID-19, require hospitalization, or die from the disease than the elderly. A combination of the viral spike (S) protein and angiotensin-converting enzyme 2 on the host cells leads to an imbalance of neurons, astrocytes, and oligodendrocytes, which makes the brain vulnerable to being an attractive target for SARS-CoV-2 infections ( Adams et al., 2020 Adams et al., 2020 ; Dhochak et al., 2020 Dhochak et al., 2020 ; Steinman et al., 2020 Steinman et al., 2020 ). Furthermore, this phenomenon can cause neurological and cognitive disorders, such as encephalitis, dementia, and neurodegeneration ( Verkhratsky et al., 2020 Verkhratsky et al., 2020 ; Conte, 2021 Conte, 2021 ; Mahalakshmi et al., 2021 Mahalakshmi et al., 2021 ; Frank et al., 2022 Frank et al., 2022 ). In screening high-risk groups in China, lung X-ray imaging is also an important way to determine whether a patient is at risk of transmission and disease severity. However, in the case of mass screening, the labor required for film reading is very large, and places high demands on imaging physicians who need experienced support. To alleviate this problem, researchers try to use computer automatic recognition to replace human resources. Among the automated detection tools provided by Alqahtani et al. (2021) Alqahtani et al. (2021) , a new method was able to detect and determine the degree of COVID-19 lung infection with 91% accuracy compared to the opinions of three experienced radiologists, and also effectively determine the severity of the disease. Rajpal et al. (2021) Rajpal et al. (2021) used computing and deep learning techniques to conduct 10-fold cross-validation and found that the overall classification accuracy of the model in identifying COVID-19 lung infections could reach 97.4%. For the first through tenth editions of the Chinese Health Commission’s Guidelines on COVID-19 Diagnosis and Treatment, please refer to Table 1 Table 1 . a Epidemiological history: 1. History of travel to or residence in Wuhan and its surrounding areas, or in other communities where cases have been reported within 14 d prior to the onset of the disease; 2. In contact with novel coronavirus-infected people (with positive results for the nucleic acid test) within 14 d prior to the onset of the disease; 3. In contact with patients who have a fever or respiratory symptoms from Wuhan and its surrounding areas, or from communities where confirmed cases have been reported within 14 d before the onset of the disease; 4. Clustered cases (two or more cases with fever and/or respiratory symptoms in a small area such as families, offices, and schools within two weeks). b Epidemiological history: 1. A history of travel or residence in the reported community within the 14 d prior to onset of illness; 2. History of contact with a novel coronavirus infection within 14 d prior to onset of illness; 3. Patients with fever or respiratory symptoms who had been in contact with communities with their own case reports within 14 d prior to onset of illness; 4. Cluster onset (more than two cases of fever and/or respiratory symptoms within 14 d in a small area such as home, office, and school class). c Clinical manifestations: 1. Fever and/or respiratory symptoms; 2. The aforementioned imaging characteristics of COVID-19; 3. Normal or decreased WBC count, normal or decreased lymphocyte count in the early stage of onset. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; RT-PCR: reverse transcription-polymerase chain reaction; Ig: immunoglobulin; COVID-19: coronavirus disease 2019; WBC: white blood cell. To be continued Edition Suspected cases Confirmed cases Changes The first edition 2020-01-15 A suspect case had been to Wuhan or had direct or indirect contact in relevant markets in Wuhan (South China Seafood Market) within two weeks prior to the onset of the disease and had typical clinical manifestations and no significant improvement or progressive aggravation after 3 d of treatment with standard antibiotics. Based on the suspected cases, the sputum, throat swab, and other respiratory tract specimens are sequenced by the whole genome of the virus, and the results are highly homologous to SARS-CoV-2. The scope of epidemiological history only focused on the Wuhan region and did not include other regions. The second edition 2020-01-22 A suspect case has any of the epidemiological history a plus all three clinical manifestations c . Suspect cases with one of the following etiological evidence: 1. Real-time fluorescent RT-PCR indicates positive for SARS-CoV-2 nucleic acid of respiratory tract specimens or blood samples. 2. Viral gene sequence is highly homologous to SARS-CoV-2. The definition of observation cases was updated as suspected cases. The third edition 2020-01-23 A suspect case needs to be identified in combination with epidemiological history a and clinical manifestations c (the first and third clinical manifestations of the second and third editions are required to be met). 3 Suspect cases with one of the aforementioned etiological evidence. The definitions of mild cases and asymptomatic cases were added. There was no imaging sign of pneumonia in mild cases, but the respiratory tract specimens positive for the etiology of SARS-CoV-2. The fourth edition 2020-01-27 A suspect case needs to be identified in combination with epidemiological history a and clinical manifestations c (the contents of epidemiological history a and clinical manifestations c are updated). The scope of epidemiological history was extended to Wuhan and its surrounding areas, or other communities with reported cases. The criteria for suspicious cases were changed to meet any two clinical manifestations. The fifth edition 2020-02-05 2 A suspect case has any of the epidemiological history a plus any two clinical manifestations c or all three clinical manifestations c if there is no clear epidemiological history. Added the diagnostic classification, clinically diagnosed cases, for Hubei Province (a suspected case with the imaging characteristics of pneumonia becomes a confirmed case when the PCR test for SARS-CoV-2 is positive). The sixth edition 2020-02-19 Suspect cases with one of the following etiological or serological evidence: 1. RT-PCR indicates positive for SARS-CoV-2. 2. Viral gene sequence is highly homologous to SARS-CoV-2. 3. Specific IgM and IgG are detectable in serum and IgG reaches a titration of at least a 4-fold increase during convalescence compared with the acute phase. The positive detection of specific IgM and IgG in serum was added as the optional criteria. The seventh edition 2020-03-04 1. A suspect case has any of the epidemiological history a plus any two clinical manifestations c or all three clinical manifestations c if there is no clear epidemiological history. 2. A suspect case has any two clinical manifestations and is positive for SARS-CoV-2 specific IgG antibodies if there is no clear epidemiological history. Suspect cases with one of the aforementioned etiological or serological evidence. None The eighth edition 2020-08-19 1. A suspect case has any of the epidemiological history b plus any two clinical manifestations c or all three clinical manifestations c if there is no clear epidemiological history. 2. A suspect case has any two clinical manifestations c and is positive for SARS-CoV-2 specific IgM antibodies if there is no clear epidemiological history b . (People who have recently received the COVID-19 vaccine do not take these conditions as reference indicators.) Suspect cases with one of the following etiological or serological evidence: 1. Tests indicate positive for SARS-CoV-2 nucleic acid. 2. Patients who have not received COVID-19 vaccines are positive for SARS-CoV-2 specific IgM and IgG antibodies. As the vaccination rate increases, attention should be paid to the exclusion of false antibody positives due to vaccination. The ninth edition 2021-03-14 1. A suspect case has any of the epidemiological history b plus any two clinical manifestations or all three clinical manifestations if there is no clear epidemiological history. 2. A suspect case has any two clinical manifestations c and is positive for SARS-CoV-2 specific IgM antibodies if there is no clear epidemiological history. (People who have recently received the COVID-19 vaccine do not take these conditions as reference indicators.) Suspect cases with one of the following etiological or serological evidence: 1. Tests indicate positive for SARS-CoV-2 nucleic acid. 2. Patients who have not received COVID-19 vaccines are positive for SARS-CoV-2 specific IgM and IgG antibodies. 3. Tests indicate positive for SARS-CoV-2 antigen. On the basis of nucleic acid testing, antigen testing is added as a supplement to further improve the ability of early case detection. The tenth edition 2023-01-05 Cancellation of suspected cases Clinical manifestations plus microbiological examination (satisfying one is enough) Microbiological examination: 1. Positive for SARS-CoV-2 nucleic acid testing; 2. Positive for SARS-CoV-2 antigen testing; 3. Positive for SARS-CoV-2 isolation and culture; 4. SARS-CoV-2-specific IgG antibody level increased to at least 4-fold level in the acute phase during recovery. The requirement of mandatory nucleic acid testing has been revoked, and any one of the microbiological tests that are conducted will suffice. 3 SARS-CoV-2 virus isolation 1 The isolation of the SARS-CoV-2 virus by cell culture contributed to diagnostic precision during the COVID-19 pandemic. This method involves the cultivation of cells in a controlled environment to support the replication and observation of the virus (da Silva et al., 2020; Harcourt et al., 2020 Harcourt et al., 2020 ; Manenti et al., 2020 Manenti et al., 2020 ; Shi P et al., 2020 Shi P et al., 2020 ). The procedure provides a critical tool for investigating the behavior of the virus and its interactions with host cells, leading to a deeper understanding of its biology and pathogenesis ( Shi R et al., 2020 Shi R et al., 2020 ). Furthermore, the method has been instrumental in the rapid development of diagnostic tests and treatments during the COVID-19 pandemic, and continues to play a key role in ongoing efforts to control and prevent the spread of this devastating virus ( Guo et al., 2021 Guo et al., 2021 ). Despite its impressive precision, the method is not without challenges. The potential for contamination is always present, requiring strict sterilization procedures and a meticulous approach to every step of the process ( Sandle, 2013 Sandle, 2013 ; Rutala and Weber, 2017 Rutala and Weber, 2017 ). The cultivation of cells and viruses in the laboratory can be a delicate balancing act, requiring precise conditions, rigorous controls, and constant monitoring ( Mackay, 2004 Mackay, 2004 ; Greenwood et al., 2012 Greenwood et al., 2012 ; Sirois, 2014 Sirois, 2014 ). Therefore, more standardized virus isolation procedures will help promote repeatability and accuracy. 4 Gene detection 1 Current COVID-19 gene detection uses mainly sequencing techniques and PCR ( Zhu et al., 2020 Zhu et al., 2020 ). SARS-CoV-2 is a virus whose genetic material is RNA. The virus’s signature sequence can be detected using direct sequencing or indirect complementary DNA (cDNA) sequencing by reverse transcription. In addition, PCR, based on the principle of complementary base pairing, can directly detect the presence of SARS-CoV-2 viral nucleic acid sequences in infected patients. 4.1 Gene sequencing 2 Next-generation sequencing (NGS) can not only provide microbiological detection and organism typing for clinical samples from patients, but also be used in epidemiology, lineage tracing, susceptibility prediction, virulence factor determination, and drug-resistance testing ( Gu et al., 2019 Gu et al., 2019 ). NGS operates by generating millions or billions of small DNA fragments from a sample and sequencing those fragments simultaneously in parallel ( Metzker, 2010 Metzker, 2010 ). The fragments are then reassembled into a complete genome using advanced computational methods ( Quail et al., 2012 Quail et al., 2012 ). In the early stages of the epidemic, in the absence of relevant information such as the SARS-CoV-2 gene sequence and primers, NGS played an important role in pathogen detection. The Chinese Center for Disease Control and Prevention isolated and sequenced the SARS-CoV-2 virus and investigated the etiology of the disease using NGS ( Chan et al., 2020 Chan et al., 2020 ; Li et al., 2020 Li et al., 2020 ; Nie et al., 2020 Nie et al., 2020 ; Zhu et al., 2020 Zhu et al., 2020 ). The virus’s complete genomic information was subsequently submitted to the World Health Organization (WHO) and stored in Global Initiative on Sharing All Influenza Data (GISAID) ( Shu and McCauley, 2017 Shu and McCauley, 2017 ; Tan et al., 2020 Tan et al., 2020 ), aiding the fight against COVID-19. To be continued NGS detection aided the development of specific RT-PCR ( Corman et al., 2020 Corman et al., 2020 ; Motayo et al., 2021 Motayo et al., 2021 ). Its results can be used to monitor variation among SARS-CoV-2 strains and provide a basis for judging a change in virulence and transmission of a mutant strain ( Bal et al., 2020 Bal et al., 2020 ; Holland et al., 2020 Holland et al., 2020 ). For example, in the new outbreak in Guangdong Province, China, in May 2021, the genetic sequencing results of infected patients were found to be highly homologous, and all were Delta variants. Furthermore, NGS can help with the development of antiviral strategies ( Dai et al., 2020 Dai et al., 2020 ) and vaccine candidates ( Kames et al., 2020 Kames et al., 2020 ), and determining the effectiveness of interventions ( Grubaugh et al., 2019 Grubaugh et al., 2019 ; Oude Munnink et al., 2020 Oude Munnink et al., 2020 ) for SARS-CoV-2. 4.2 PCR 2 As a technique for amplifying nucleic acid in vitro with high sensitivity and specificity for early diagnosis, PCR is regarded as the “gold standard” for detecting the presence of viruses. In addition to traditional RT-PCR methods, new techniques are being developed such as isothermal and thermostatic amplification chips, which can achieve higher operational convenience and a shorter detection time. Table 2 Table 2 summarizes and compares several nucleic-based molecular biological detection methods, serving as a resource for increasingly complex detection occasions and requirements. RT-PCR: reverse transcription-polymerase chain reaction; cDNA: complementary DNA; COVID-19: coronavirus disease 2019; LAMP: loop-mediated isothermal amplification; CRISPR: clustered regularly interspaced short palindromic repeats; Cas12a: CRISPR-associated protein 12a system; gRNA: guide RNA; RNP: RNA polymerase; qPCR: quantitative PCR. Method Principle Time Advantages Disadvantages Evaluation Samples References RT-PCR Reverse transcriptase converts the viral RNA genome to cDNA. The DNA copy of the virus's genetic material grows exponentially after repeated cycles of amplification, making it easy to detect. 2 h 1. Low cost; 2. Good specificity; 3. High sensitivity; 4. Can be used for early diagnosis. 1. It takes a long time and the operation is complicated; 2. False-negative is easy to occur due to the influence of multiple factors; 3. It has high personnel and laboratory equipment requirements, making it difficult to popularize in primary medical institutions. It is the most widely used and one of the "gold standards" for diagnosis. It can be used in a variety of situations, including case diagnosis, condition analysis, and population screening. Blood, tissue, and cell lines Lanciotti et al., 1992 Lanciotti et al., 1992 ; Gill et al., 2009 Gill et al., 2009 ; Grubaugh et al., 2019 Grubaugh et al., 2019 ; Kames et al., 2020 Kames et al., 2020 ; Oude Munnink et al., 2020 Oude Munnink et al., 2020 ; Tsang et al., 2021 Tsang et al., 2021 Isothermal amplification technique For each region of the target gene, 4‒6 pairs of specific primers were designed. The nucleic acid was then amplified by incubating them with reverse transcriptase and DNA polymerase with strong chain replacement activity and high-temperature tolerance for 20 to 60 min at a constant temperature. 15‒60 min 1. High sensitivity; 2. Quick and portable; 3. Results visualization. The possibility of non-specific binding of primers is high, resulting in false positives. Used to monitor a large number of exposed individuals and to facilitate screening in hospitals and the public sector. Saliva, urine, and sputum Liu et al., 2016 Liu et al., 2016 ; Li et al., 2018; Gao W et al., 2019 Gao W et al., 2019 ; Pumford et al., 2020 Pumford et al., 2020 ; Smyrlaki et al., 2020 Smyrlaki et al., 2020 ; Sun and Guan, 2020 Sun and Guan, 2020 ; Gao YP et al., 2022 Gao YP et al., 2022 Isothermal amplification chip method Combining isothermal amplification technology with microfluidic chip technology. 50 min 1. Simultaneous detection of multiple pathogens from the same sample; 2. Strong integration ability and a high degree of automation; 3. Detection time is so short that suitable for real-time detection. Chip design, material selection, processing, packaging, and storage are all challenging tasks. Multiple indicators can be combined to effectively distinguish influenza patients from COVID-19 patients and achieve an accurate patient diagnosis. Saliva, urine, and sputum Gill et al., 2009 Gill et al., 2009 ; Pumford et al., 2020 Pumford et al., 2020 ; Smyrlaki et al., 2020 Smyrlaki et al., 2020 ; Sun and Guan, 2020 Sun and Guan, 2020 ; Tsang et al., 2021 Tsang et al., 2021 ; Wang et al., 2021 Wang et al., 2021 ; Gao YP et al., 2022 Gao YP et al., 2022 LAMP detection based on CRISPR/Cas12a The target sequence was amplified. To activate Cas12a, RNA was directed to recognize the target sequence and interact with the Cas12a-gRNA RNP complex. As a result, the target sequence is specifically cut and produces fluorescence. 40 min 1. Reduced operational complexity and the possibility of cross-contamination; 2. Strong system stability; 3. Low requirements on operating environment, and can be used for field testing. The development began relatively late, and further research is required to ensure detection accuracy. This method is in good agreement with qPCR and provides a simple and reliable field diagnosis method suitable for community testing. Bacterial cultures, environmental samples, and clinical specimens Li YY et al., 2017 Li YY et al., 2017 ; Hu et al., 2020 Hu et al., 2020 ; Yan et al., 2020 Yan et al., 2020 ; Zhang et al., 2020 Zhang et al., 2020 ; Li F et al., 2021 Li F et al., 2021 4.2.1　RT-PCR 3 RT-PCR technology converts the virus’s RNA genome into cDNA, using RNA-dependent DNA polymerase (reverse transcriptase). The DNA copy of the virus genetic material increases exponentially after repeated cyclic amplification, making detection easier. This cDNA is then used as a template for the PCR, where the DNA is amplified millions of times over, allowing for highly sensitive detection ( Lanciotti et al., 1992 Lanciotti et al., 1992 ). The beauty of RT-PCR lies in its ability to detect RNA molecules rapidly and accurately, even at very low concentrations ( Iscove et al., 2002 Iscove et al., 2002 ). The design of virus-specific primers and probes accelerated following the release of the SARS-CoV-2 genome sequence ( Yuan et al., 2021 Yuan et al., 2021 ). As reported by Pumford et al. (2020) Pumford et al. (2020) , the process begins with the design of specific primers, which serve as the foundation for the amplification of the viral RNA present in a sample. These primers are then combined with probes, often labeled with fluorescent dyes, to enable the detection of the amplified products and to quantify the amount of virus present. The virus’s RNA sequence serves as a highly specific RT-PCR marker, distinguishing it from other pathogens. The design of primers and probes for specific gene detection has greatly improved the technology’s sensitivity. Considering the positive rates of various specimens and the degree of acceptance by the subjects, the following are recommended: (1) Nasopharyngeal swabs should be taken as a priority for screening of asymptomatically infected persons and the general population. (2) For patients with acute COVID-19, respiratory tract samples should be taken. (3) At a later stage, additional fecal samples can be collected for monitoring recovery from the disease. (4) False negatives can be effectively avoided by combining samples from different sources. In conclusion, RT-PCR is regarded as the best diagnostic option for a wide range of surveillance strategies. There are many available RT-PCR kits based on one-step amplification, which ensures the standardized process and low systematic error ( Smyrlaki et al., 2020 Smyrlaki et al., 2020 ; Wang CJ et al., 2022 Wang CJ et al., 2022 ). This method, however, has a certain false-negative rate due to non-standard sampling procedures or low viral loads ( Sun and Guan, 2020 Sun and Guan, 2020 ). In the laboratory testing technology guide of the COVID-19 Diagnosis and Treatment Guideline (the fifth edition), the National Health Commission clearly states that a negative PCR test result cannot exclude SARS-CoV-2 infection, and that it is necessary to exclude the factors that may produce a false negative. To be continued 4.2.2　Isothermal amplification technique 3 Isothermal amplification is a new and simple nucleic acid amplification technology. In contrast to RT-PCR, the reaction process is kept at a constant temperature, eliminating the need for temperature cycling, and rapid nucleic acid amplification can be accomplished by adding active enzymes and specific primers. By leveraging the use of heat-stable enzymes, such as recombinase polymerase amplification (RPA) and ligase chain reaction (LCR), this method has shown remarkable efficiency and specificity in detecting and quantifying low-abundance target sequences ( Li et al., 2017 Li et al., 2017 ; Gao YP et al., 2022 Gao YP et al., 2022 ). The requirements of isothermal amplification for specialized instruments are greatly simplified and such instruments may even not be needed, and the reaction time is greatly reduced, allowing the technique to better meet the needs of rapid and simple detection. Currently, the most widely used isothermal amplification technique is loop-mediated isothermal amplification (LAMP), whose main advantage is the ability to visualize the results. It can be used for quantitative analysis based on turbidity and color changes. Because LAMP performs the amplification reaction at a lower temperature, the possibility of non-specific binding of primers increases, which makes false positives more likely. Fortunately, detection specificity can be improved by combining fluorescent probes and designing appropriate primers. Yan et al. (2020) Yan et al. (2020) designed five groups of primers based on the reverse transcription-LAMP (RT-LAMP) method, among which the open reading frames 1ab (ORF1ab)‍-4 and S-123 primers amplified the gene in the shortest time. Hu et al. (2020) Hu et al. (2020) confirmed that RT-LAMP had a higher sensitivity and specificity in the diagnosis of SARS-CoV-2 infection than RT-qPCR. Therefore, the RT-LAMP detection and LAMP sequencing can broaden the range of available test methods and supplement RT-PCR-based single and combination tests with faster, simpler, and potentially less expensive test methods. The isothermal amplification chip method, combining isothermal amplification technology with microfluidic chip technology, can be used to detect multiple pathogens in the same sample. It is highly integrated with a high degree of automation, short detection time, and low detection limit (up to 100 copies/mL). 4.2.3　LAMP detection based on CRISPR/Cas12a 3 Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a nucleic acid testing tool with a high degree of specificity. The CRISPR/CRISPR-associated protein 12a system (Cas12a)-based LAMP combines RT-LAMP with CRISPR/Cas12a, and is a groundbreaking approach to disease diagnosis. An RT-LAMP reaction is carried out at 65 ℃ to amplify the SARS-CoV-2-specific nucleic acid sequence, and detection is then performed at 37 ℃ using CRISPR/Cas12a technology. This system works using a guide RNA (gRNA) to direct the Cas12a enzyme to a target DNA sequence, where it cleaves the DNA. In the LAMP detection method, the target DNA is amplified in an isothermal reaction and the resulting amplicons are then detected using specific fluorescent dyes. The entire amplification and detection process is completed within 40 min ( Chen et al., 2020 Chen et al., 2020 ). This method is highly specific and sensitive, avoiding cross-reactivity with related coronaviruses. Pang et al. (2020) Pang et al. (2020) developed a single-tube method for detecting SARS-CoV-2 in patients, combining the benefits of RT-LAMP isothermal exponential amplification and the sequence recognition specificity of the CRISPR/Cas12a system, which further simplifies operation and reduces contamination at a constant temperature. Wang et al. (2021) Wang et al. (2021) proposed the “opvCRISPR” detection system, which combines RT-LAMP amplification and CRISPR cutting. This system has a high specificity, a sensitivity similar to that of RT-PCR, and short detection time, and requires less equipment. Thus, it compensates for the shortcomings of RT-PCR, which is not suitable for large-scale diagnosis, requires expensive equipment, and has a long reaction time. The opvCRISPR method has enormous potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics ( Wang et al., 2021 Wang et al., 2021 ). 5 Immunodiagnosis 1 Serological tests are quick, simple, sensitive, accurate, and stable, making them ideal for large-scale testing and tracing previously infected patients ( Dheda et al., 2013 Dheda et al., 2013 ; Mercer and Salit, 2021 Mercer and Salit, 2021 ). There are numerous serological detection methods for SARS-CoV-2, including direct detection and indirect detection. Direct detection involves detecting SARS-CoV-2 RNA sequences or landmark proteins like the S protein ( Mohammadi et al., 2022 Mohammadi et al., 2022 ). Antibody detection, which looks for SARS-CoV-2-specific antibodies in the body, is an example of indirect detection. Immunoglobulin G (IgG), IgM, or both antibodies were used to test for COVID-19 (la Marca et al., 2020; Safiabadi Tali et al., 2021 Safiabadi Tali et al., 2021 ). The National Health Commission included the SARS-CoV-2 serum antibody test as an auxiliary detection method in the Protocol on Prevention and Control of COVID-19 (Edition 6) ( http://en. http://en. ‍nhc.‍gov.‍cn/2020-03/29/c_78468.‍htm). Regarding the definition of specific IgM and IgG positivity, IgM or IgG changes from negative to positive, or IgM or IgG has a titer of at least 4-fold increase during convalescence compared with the acute phase. The rapid and sensitive immunological test method is being promoted as an auxiliary test method for the rapid diagnosis of SARS-CoV-2 across the country. It can reduce the false-negative rate when performed in conjunction with a PCR test, track the progression of the disease, and reduce the risk of exposure of medical personnel due to the ease of sampling ( Chau et al., 2020 Chau et al., 2020 ; Kucirka et al., 2020 Kucirka et al., 2020 ). 5.1 Antigen detection 2 Antigen detection has high accuracy and a fast reporting speed (usually about half an hour). It can also provide results at an earlier stage of infection ( Santiago, 2020 Santiago, 2020 ; Yüce et al., 2021 Yüce et al., 2021 ). Rapid antigen testing can identify SARS-CoV-2 positive individuals by detecting the SARS-CoV-2 nucleocapsid protein or S protein in swabs taken from the upper respiratory tract of suspected infected subjects ( Taleghani and Taghipour, 2021 Taleghani and Taghipour, 2021 ). A lateral flow test employs the immunochromatographic principle: the test sample is moved across the matrix to bind antigens to antibodies through capillary action, allowing these complexes to be detected and visualized ( Singh et al., 2015 Singh et al., 2015 ; Koczula and Gallotta, 2016 Koczula and Gallotta, 2016 ). Antigen detection is not only important for diagnosing infected patients, but also can reduce the reliance on the PCR assay to some extent ( Singh et al., 2015 Singh et al., 2015 ). The sensitivity of rapid point-of-care antigen detection for SARS-CoV-2 infection within 7 d of symptom onset ranged from 77.3% (duration 1 to 33 d) to 100% in a low-prevalence setting ( Chen et al., 2021 Chen et al., 2021 ; Muhi et al., 2021 Muhi et al., 2021 ). Corman et al. (2021) Corman et al. (2021) and Hirotsu et al. (2021a Hirotsu et al. (2021a , 2021b) 2021b) recently comprehensively evaluated PCR-based quantitative antigen tests and found that the Roche and Lumipulse antigen tests showed high concordance up to 9 d after symptom onset using RT-PCR as a reference. The Lumipulse ® antigen assay has a sensitivity and specificity of over 90% in RT-PCR positive samples, demonstrating that the antigen detection system can be safely used for screening in the general population ( Gili et al., 2021 Gili et al., 2021 ). When it comes to detecting recombinant proteins or viruses, the RapiGEN test outperforms all others. Krüttgen et al. (2021) Krüttgen et al. (2021) researched the sensitivity and specificity of the SARS-CoV-2 Rapid Antigen Test (Roche) using swabs from patients previously tested by SARS-CoV-2 PCR and concluded that the sensitivity and specificity of the antigen assay are inferior to those of a PCR assay. A study found that immunochromatographic AMP rapid antigen test was highly sensitive at low cycle threshold ( C T ) values when rapid RT-PCR tests were not feasible, making it a valuable tool for front-line testing ( Leixner et al., 2021 Leixner et al., 2021 ). All these studies suggest that antigen tests are somewhat feasible, but may be limited by viral load. The sensitivity and specificity of currently approved antigenic assays are not as good as those of PCR assays, but antigenic assays may provide an alternative that is quick and easy to perform ( García-Fiñana and Buchan, 2021 García-Fiñana and Buchan, 2021 ). Their accuracy is heavily reliant on a high viral load ( Menchinelli et al., 2021 Menchinelli et al., 2021 ), which means that positive results are more likely in the early stages of the disease. This also explains why the LUMIPULSE G600II automated immunoassay analyzer (FujireBio) had a sensitivity of only 55% when applied to long-term follow-up samples from a small number of patients ( Hirotsu et al., 2020 Hirotsu et al., 2020 ). According to the WHO interim guidelines, negative predictive value (NPV) is more reliable in populations with low infection rates, whereas a higher positive predictive value (PPV) can reduce the time burden and cost of procalcitonin (PCT) in populations with high infection rates. Therefore, we believe that attention should be paid to how antigen detection and PCR tests should be allocated in the diagnosis of COVID-19 patients with different infection ratios, infection tolerance, and detection conditions in different regions ( Mina et al., 2020 Mina et al., 2020 ; Fitzpatrick et al., 2021 Fitzpatrick et al., 2021 ). When the viral load is reduced to a concentration unlikely to be detectable by antigen-rapid diagnostic testing (Ag-RDT) in patients going to hospital more than seven days after symptom onset, the best option is to confirm COVID-19 using a combination of molecular and antibody tests ( Peeling et al., 2021 Peeling et al., 2021 ). More notably, recent studies have shown that some antigen tests are also good at identifying mutated viruses. Soni et al. (2022) Soni et al. (2022) compared the sensitivity of Ag-RDT to the Delta and Omicron variants and found that the sensitivity was highly consistent. Although certain mutated sites affected the accuracy of some antigen tests, other tests were unaffected when comparing results for antigen-negative samples identified by Bourassa et al. (2021) Bourassa et al. (2021) using different tests. Therefore, careful screening and cross-use of different antigen detection methods could reduce the probability of missed diagnosis. The development of antigen detection deserves further attention as its accuracy still lags a little behind the gold standard. Whether a detection method is worthy of research and large-scale implementation depends not only on the accuracy and sensitivity of the method. A test method for COVID-19 is always intended to be used on patients or the public, and its operability on a large scale must also be considered. The operational difficulty of antigen detection is very low and the result interpretation is relatively simple. These advantages are very important in large-scale deployment. Therefore, further development of antigen detection methods is desirable. 5.2 Antibody detection 2 Antibody detection is now widely used in clinical detection and is included in diagnostic evidence along with RT-PCR ( Li et al., 2020 Li et al., 2020 ). However, antibody detection cannot replace PCR detection and viral gene sequencing as the gold standard for confirming SARS-CoV-2 infection ( Lv et al., 2020 Lv et al., 2020 ). On the one hand, serological detection can only indirectly prove the existence of the virus ( Peto, 2020 Peto, 2020 ). On the other hand, at the stage of large-scale vaccination, it is unclear whether the positive antibody is the result of SARS-CoV-2 infection or vaccination ( Yue et al., 2022 Yue et al., 2022 ). This rapid, simple, and highly sensitive method can improve the sensitivity of the diagnosis of COVID-19 and represents an appropriate supplement to a PCR test ( Wu et al., 2022 Wu et al., 2022 ). The main methods of antibody detection include chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and lateral flow immunoassay (LFIA) ( Nicol et al., 2020 Nicol et al., 2020 ). Montesinos et al. (2020) Montesinos et al. (2020) evaluated CLIA, ELISA, and LFIA, and pointed out that they were accurate and equivalent in detecting SARS-CoV-2 antibodies. This confirmed their suitability for clinical use and for formulating an epidemiological strategy for the COVID-19 pandemic ( Montesinos et al., 2020 Montesinos et al., 2020 ; Yadav et al., 2021 Yadav et al., 2021 ). Antibody detection is mainly for IgM and IgG antibodies. Li et al. (2020) Li et al. (2020) used a lateral immunoassay to simultaneously detect IgM and IgG antibodies against SARS-CoV-2 in human blood within 15 min. They found that this antibody detection method has high sensitivity and specificity, and that IgM-IgG combined detection has better practicability and sensitivity than IgM or IgG detection alone ( Li et al., 2020 Li et al., 2020 ). The change of antibody titer in serum provides a very important reference value for the judgment of the course of infection and prognosis. According to Wu et al. (2020) Wu et al. (2020) , the presence of antibodies in COVID-19 patients was < 40% within one week after onset and rapidly increased to 100.0% (antibody), 94.3% (IgM), and 79.8% (IgG) by Day 15. In contrast, RNA detection decreased from 66.7% (58/87) in samples collected prior to Day 7 to 45.5% (25/55) during Days 15‍‒‍39 ( Wu et al., 2020 Wu et al., 2020 ). The dynamic monitoring of antibodies can improve understanding of the rules of antibody production and play an important role in follow-up investigations of the COVID-19 epidemic, the development of antibody drugs and vaccines, and clinical diagnosis and treatment. Moreover, it can be more effective in preventing and controlling COVID-19 ( Qiu et al., 2020 Qiu et al., 2020 ; Wang XN et al., 2020 Wang XN et al., 2020 ). If the SARS-CoV-2 antibody detection is combined with the PCR test to interpret the results of the “nucleic acid+antibody” test, it will be effective for improving the detection rate of diseases, monitoring diseases and controlling the epidemic situation, and achieving “early detection, early diagnosis, and early treatment.” Table 3 Table 3 summarizes the different possible results and diagnostic significance of coordinated nucleic acid and antibody testing. Table 4 Table 4 summarizes the current methods commonly used for antibody detection. The data are referenced from the studies by Kubina and Dziedzic (2020) Kubina and Dziedzic (2020) , Fang et al. (2021) Fang et al. (2021) , Yan et al. (2021) Yan et al. (2021) , and Dou et al. (2022) Dou et al. (2022) . SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; Ig: immunoglobulin. Result IgM IgG Diagnostic significance 4 Positive (+) + + The virus is active, but the body has a certain immunity. + - In the early stage of infection, IgG is not present or have low. - + Be in the middle or late stage of infection or have recurrent infection. - - In the window period of infection, no antibody has been detected yet. 4 Negative (-) + + Be in recovery, but still have a certain number of antibodies. + - Most likely in the infection period, nucleic acid should be retested. - + Have recovered from a previous infection. - - There was no infection and no protective antibodies. LFIA: lateral flow immunoassay; ELISA: enzyme-linked immunosorbent assay; CLIA: chemiluminescent immunoassay. Method Sensitivity(%) Specificity(%) Time Advantages Disadvantages LFIA 71.4‒88.7 90.6‒98.4 15‒20 min 1. Small and portable; 2. Quick and sensitive at a low cost; 3. Fingertip blood can be used for testing. 1. Quantitative analysis is unavailable; 2. It is easy to be contaminated by other factors and lead to false positives. ELISA 75.6‒100.0 85.7‒100.0 1‒5 h 1. High specificity and sensitivity; 2. Low difficulty in carrier standardization; 3. Low requirements for equipment. 1. Time-consuming and complicated; 2. Only semi-quantitative detection. CLIA 96.0‒98.0 89.4‒100.0 1‒2 h 1. High sensitivity and time-saving; 2. No enzymes; 3. Capable of quantitative analysis and automated high-throughput detection. 1. Special equipment is required; 2. High cost. Protein microarray 1. Photosensitive dye marking; 2. Less specimen consumption but high sensitivity; 3. High throughput and parallel analysis. 1. High requirements for equipment; 2. Possibility of false positives. The COVID-19 Diagnosis and Treatment Guideline (the seventh edition) adds a serological test method and states explicitly that specific antibody testing, PCR testing, and viral gene sequencing should be taken together as diagnostic evidence. In this case, the serological test results can not only be used to monitor the immune level, but also be a further supplement to the PCR test result. Compared to PCR, serological detection has advantages like having a simple operation, simple sample collection, and low exposure risk. Additionally, monitoring of SARS-CoV-2 antibodies using dried blood spots (DBSs) is a cutting-edge innovation in the fight against the COVID pandemic. This test can be operated by any individual given that it requires only dried blood samples pricked from fingers. In a study using this method to evaluate sensitivity and specificity based on 31 SARS-CoV-2 infected patients and 80 healthy volunteers, Karp et al. (2020) Karp et al. (2020) and Beyerl et al. (2021) Beyerl et al. (2021) stated that the technique perfectly distinguished positive and negative cases, and both the sensitivity and specificity reached almost 100%. This method allows for a simple, non-invasive, and cost-effective way to measure the presence of antibodies in an individual’s blood, and also may be a convenient protocol for school age children or the disabled ( Karp et al., 2020 Karp et al., 2020 ; Beyerl et al., 2021 Beyerl et al., 2021 ). By using DBS, researchers can easily collect and store blood samples, eliminating the need for complicated laboratory procedures ( Beyerl et al., 2021 Beyerl et al., 2021 ; Miesse et al., 2022 Miesse et al., 2022 ). In other studies by McDade et al. (2020) McDade et al. (2020) and Morley et al. (2020) Morley et al. (2020) , DBS analysis was proven to be a highly reliable and accurate method for detecting SARS-CoV-2 antibodies. Gaugler et al. (2021) Gaugler et al. (2021) improved detection throughput using an automatic DBS processing and extracting technique. This technique, which greatly reduces detection cost and supports at-home testing, also demonstrates the potential of DBS for widespread sero-surveillance and tracking the evolution of the pandemic ( Zava and Zava, 2021 Zava and Zava, 2021 ). Though there have been few studies involving DBS analysis in China, especially in special populations such as school age children, it has great potential for use in the post-pandemic era. Note that regardless of the method, serological test results provide only circumstantial evidence, which cannot be used as confirmed diagnostic evidence. 6 Imaging diagnosis 1 In an emergency, laboratory testing techniques may not be appropriate, and RT-PCR may produce a false negative if the viral load is insufficient. According to some studies, computed tomography (CT) scanning has higher sensitivity (88%‍‒‍98%) and a lower false-negative rate than RT-PCR ( Fang et al., 2020 Fang et al., 2020 ). In a study of 1014 patients, the sensitivity of chest CT scans for COVID-19 diagnosis was 97% ( Xu et al., 2020 Xu et al., 2020 ). With analysis of serial RT-PCR assays and CT scans, the mean interval between the initial negative to positive RT-PCR results was about 5 d, indicating that CT scans could play a key role in the early detection and treatment of COVID-19. Because of the high sensitivity of imaging detection, the COVID-19 Diagnosis and Treatment Guideline (the fifth edition) added clinically diagnosed cases for Hubei Province, China. Cases with suspected pneumonia based on imaging characteristics but negative PCR test results were included in the scope of confirmed cases for unified isolating diagnosis and treatment. The presence of ground-glass opacity (GGO) in the peri-pulmonary and subpleural areas is the most notable CT feature of COVID-19 pneumonia ( Wang YH et al., 2020 Wang YH et al., 2020 ). The characteristics change over time, depending on the stage and severity of the pulmonary infection ( Kobayashi and Mitsudomi, 2013 Kobayashi and Mitsudomi, 2013 ). The imaging stages and manifestations of COVID-19 are summarized in Table 5 Table 5 . COVID-19: coronavirus disease 2019; GGO: ground-glass opacity. Period Imaging findings Early period In the first week of onset, the thin ground glass density shadow is common. The thickening of the bronchial vascular bundle can be seen, or with local gridded thickening of the interlobular septum. Exacerbation The range of GGO shadows increases with increased density. Some are fused into lobules, while others are widely fused into bands or large sheets of dense shadows, with visible and multiple consolidations. Critical period Diffuse gridded GGO shadows appeared in both lungs, and a few individuals showed "white lungs." The majority of the lesions consisted of small flake consolidation. Both pleural cavities may have a small effusion. Absorption The lesion's scope and density were reduced. The GGO shadow may completely disappear as the exudate is gradually absorbed. Some patients, however, may have residual pulmonary fiber strip shadow. Pan et al. (2020) Pan et al. (2020) investigated changes in the lung over time in patients recovering from COVID-19. They discovered that pulmonary involvement gradually decreased 10 d after symptom onset and then increased, showing mainly imaging signs such as GGO, paving stone signs and consolidation shadows. These symptoms then subsided 14 d after they first appeared. Bernheim et al. (2020) Bernheim et al. (2020) discovered a high frequency (56%) of normal CT results in the early stages of disease (0‒2 d), with the most severe pulmonary infection peaking about 10 d after symptom onset. Tsang et al. (2021) Tsang et al. (2021) studied longitudinal changes and confirmed that isolated GGO was most common after symptom onset, and that a mixed pattern of GGO and irregular linear opacity peaked from 6 to 11 d of the disease. Notably, the main limitation of CT in confirming COVID-19 is its low specificity (25%). Due to imaging features that may overlap, it is difficult to distinguish COVID-19 from other viral pneumonias ( Bai et al., 2020 Bai et al., 2020 ). Previous diagnostic data from radiology departments at several hospitals in Hubei Province, China, showed that 30% to 40% of patients with COVID-19 symptoms based on chest CT had negative PCR test results. CT detection has a higher positive rate than nucleic acid detection, which allows medical staff to make more accurate clinical diagnoses. Furthermore, CT detection can help guide clinical decision-making and provide prognostic information. 7 Authors’ opinion 1 Since the start of the pandemic, the requirements for the quality and speed of sample detection have increased. To some extent, this has promoted progress in detection technologies in China and increased the level of attention to detection. In addition, rapid and accurate testing has been proven to be a key component of the fight against COVID-19. At the current stage of the pandemic development in China, it is almost impossible for a large-scale epidemic to occur under the strict prevention and control of the government, the united efforts of grassroots organizations, and the meticulous cooperation of the entire nation ( Cheng et al., 2023 Cheng et al., 2023 ). However, due to the continual evolution of the virus and the emergence of individuals who remain positive after sitting through the formal quarantine period, a small number of sporadic cases cannot be completely avoided. Nationwide testing can be performed occasionally, but requires a lot of labor and materials, which depletes available medical resources. At the moment, it is critical to distinguish between healthy and infectious patients swiftly and accurately. Therefore, the most important current aim is to improve detection efficiency and accuracy while keeping detection methods simple. Further, to minimize the risk of infection and prevent the spread of the virus, it is crucial to adhere to strict biosafety and biosecurity measures in the laboratory. These measures include wearing personal protective equipment, implementing decontamination procedures, and following standard operating procedures (SOPs) for handling the virus. Based on the detection methods described above, the main diagnosis method continues to rely on a combination of antigen and antibody detection, RT-PCR, and clinical symptoms, each with its own set of advantages and disadvantages. In the case of antigen detection, although the detection time is very short, it has certain viral load requirements, which may mean that the patient is already infectious by the time the virus reaches detectable levels. Although the amplification used in RT-PCR can greatly improve accuracy and the technology has advanced significantly, RT-PCR still cannot match the reporting speed of antigen detection, due to the limits of its concept. Therefore, we believe that the main focus should be on further improving the speed and accuracy of RT-PCR detection. On the other hand, there is also a need to find alternative techniques that can replace RT-PCR as the gold standard for confirming SARS-CoV-2 infection. By raising the sensitivity of antigen detection to a level comparable to that of RT-PCR and considering aspects such as the detection principle, a rapid and accurate detection approach with low environmental requirements may be created. Recent research has indicated that nanomaterials such as graphene, or related antigen detection technologies, can considerably enhance accuracy. Thus, the viral load requirement can be minimized and the utilization rate of antigen testing can be improved in China. Zhang et al. (2022) Zhang et al. (2022) used a graphene mixture to boost the sensitivity of interleukin-6 (IL-6) detection. Wang YW et al. (2022) Wang YW et al. (2022) created an electrochemical immunosensor based on staphylococcal protein A (SPA) and reduced graphene oxide-polyethyleneimine-silver nanoparticles-Nafion (rGO-PEI-Ag-Nf) for rapid detection of mAbs. Kuntip et al. (2021) Kuntip et al. (2021) discovered that graphene quantum dots have a high capacity for microRNA (miRNA) adsorption. In other words, graphene shows a higher adsorption capacity for the detection of particular biological macromolecules, which improves the sensitivity of detection and reduces the detection threshold. Gao JW et al. (2022) Gao JW et al. (2022) have developed a field-effect transistor biosensor based on a graphene oxide (GO)/graphene van der Waals heterostructure for selective and ultra-sensitive SARS-CoV-2 protein detection. It can effectively improve the fixed density of SARS-CoV-2 captured antibody, with strong selectivity and high sensitivity, and represents a potential method for rapid and accurate detection of SARS-CoV-2. To some extent, graphene has been shown to improve the sensitivity of biological macromolecule detection. Extending this technique’s application to the rapid identification of COVID-19 is a very promising research direction, especially in the application of fast antigen detection. References Adams SH , Park MJ , Schaub JP , et al. , 2020 . Medical vulnerability of young adults to severe COVID-19 illness—data from the national health interview survey . J Adolesc Health , 67 ( 3 ): 362 - 368 . https://doi.org/10.1016/j.jadohealth.2020.06.025 https://doi.org/10.1016/j.jadohealth.2020.06.025 Medical vulnerability of young adults to severe COVID-19 illness—data from the national health interview survey Alqahtani MS , Abbas M , Alqahtani A , et al. , 2021 . A novel computational model for detecting the severity of inflammation in confirmed COVID-19 patients using chest X-ray images . Diagnostics (Basel) , 11 ( 5 ): 855 . https://doi.org/10.3390/diagnostics11050855 https://doi.org/10.3390/diagnostics11050855 A novel computational model for detecting the severity of inflammation in confirmed COVID-19 patients using chest X-ray images Al-Tawfiq JA , 2020 . Asymptomatic coronavirus infection: MERS-CoV and SARS-CoV-2 (COVID-19) . Travel Med Infect Dis , 35 : 101608 . https://doi.org/10.1016/j.tmaid.2020.101608 https://doi.org/10.1016/j.tmaid.2020.101608 Asymptomatic coronavirus infection: MERS-CoV and SARS-CoV-2 (COVID-19) Bai HX , Hsieh B , Xiong Z , et al. , 2020 . Performance of radiologists in differentiating COVID-19 from non-COVID-19 viral pneumonia at chest CT . Radiology , 296 ( 2 ): E46 - E54 . https://doi.org/10.1148/radiol.2020200823 https://doi.org/10.1148/radiol.2020200823 Performance of radiologists in differentiating COVID-19 from non-COVID-19 viral pneumonia at chest CT Bai Y , Tao XN , 2021 . Comparison of COVID-19 and influenza characteristics . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 2 ): 87 - 98 . https://doi.org/10.1631/jzus.B2000479 https://doi.org/10.1631/jzus.B2000479 Comparison of COVID-19 and influenza characteristics Bal A , Destras G , Gaymard A , et al. , 2020 . Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals an amino acid deletion in nsp2 (Asp268del) . Clin Microbiol Infect , 26 ( 7 ): 960 - 962 . https://doi.org/10.1016/j.cmi.2020.03.020 https://doi.org/10.1016/j.cmi.2020.03.020 Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals an amino acid deletion in nsp2 (Asp268del) Bernheim A , Mei XY , Huang MQ , et al. , 2020 . Chest CT findings in coronavirus disease-19 (COVID-19): relationship to duration of infection . Radiology , 295 ( 3 ): 685 - 691 . https://doi.org/10.1148/radiol.2020200463 https://doi.org/10.1148/radiol.2020200463 Chest CT findings in coronavirus disease-19 (COVID-19): relationship to duration of infection Beyerl J , Rubio-Acero R , Castelletti N , et al. , 2021 . A dried blood spot protocol for high throughput analysis of SARS-CoV-2 serology based on the Roche Elecsys anti-N assay . eBioMedicine , 70 : 103502 . https://doi.org/10.1016/j.ebiom.2021.103502 https://doi.org/10.1016/j.ebiom.2021.103502 A dried blood spot protocol for high throughput analysis of SARS-CoV-2 serology based on the Roche Elecsys anti-N assay Bourassa L , Perchetti GA , Phung Q , et al. , 2021 . A SARS-CoV-2 nucleocapsid variant that affects antigen test performance . J Clin Virol , 141 : 104900 . https://doi.org/10.1016/j.jcv.2021.104900 https://doi.org/10.1016/j.jcv.2021.104900 A SARS-CoV-2 nucleocapsid variant that affects antigen test performance Brodin P , 2021 . Immune determinants of COVID-19 disease presentation and severity . Nat Med , 27 ( 1 ): 28 - 33 . https://doi.org/10.1038/s41591-020-01202-8 https://doi.org/10.1038/s41591-020-01202-8 Immune determinants of COVID-19 disease presentation and severity Cao YL , Jian FC , Wang J , et al. , 2023 . Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution . Nature , 614 ( 7948 ): 521 - 529 . https://doi.org/10.1038/s41586-022-05644-7 https://doi.org/10.1038/s41586-022-05644-7 Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution Chakraborty S , Chandran D , Mohapatra RK , et al. , 2022 . Langya virus, a newly identified Henipavirus in China‍‒Zoonotic pathogen causing febrile illness in humans, and its health concerns: current knowledge and counteracting strategies‒Correspondence . Int J Surg , 105 : 106882 . https://doi.org/10.1016/j.ijsu.2022.106882 https://doi.org/10.1016/j.ijsu.2022.106882 Langya virus, a newly identified Henipavirus in China‍‒Zoonotic pathogen causing febrile illness in humans, and its health concerns: current knowledge and counteracting strategies‒Correspondence Chan JF , Yuan SF , Kok KH , et al. , 2020 . A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster . Lancet , 395 ( 10223 ): 514 - 523 . https://doi.org/10.1016/S0140-6736(20)30154-9 https://doi.org/10.1016/S0140-6736(20)30154-9 A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Chandran D , Dhama K , Chakraborty S , et al. , 2022 . Monkeypox: an update on current knowledge and research advances . J Exp Biol Agric Sci , 10 ( 4 ): 679 - 688 . https://doi.org/10.18006/2022.10(4).679.688 https://doi.org/10.18006/2022.10(4).679.688 Monkeypox: an update on current knowledge and research advances Chau CH , Strope JD , Figg WD , 2020 . COVID-19 clinical diagnostics and testing technology . Pharmacotherapy , 40 ( 8 ): 857 - 868 . https://doi.org/10.1002/phar.2439 https://doi.org/10.1002/phar.2439 COVID-19 clinical diagnostics and testing technology Chen XP , Hu WJ , Yang M , et al. , 2021 . Risk factors for the delayed viral clearance in COVID-19 patients . J Clin Hypertens (Greenwich) , 23 ( 8 ): 1483 - 1489 . https://doi.org/10.1111/jch.14308 https://doi.org/10.1111/jch.14308 Risk factors for the delayed viral clearance in COVID-19 patients Chen YJ , Shi Y , Chen Y , et al. , 2020 . Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: a promising method in the point-of-care detection . Biosens Bioelectron , 169 : 112642 . https://doi.org/10.1016/j.bios.2020.112642 https://doi.org/10.1016/j.bios.2020.112642 Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: a promising method in the point-of-care detection Cheng ZJ , Shan J , 2020 . 2019 Novel coronavirus: where we are and what we know . Infection , 48 ( 2 ): 155 - 163 . https://doi.org/10.1007/s15010-020-01401-y https://doi.org/10.1007/s15010-020-01401-y 2019 Novel coronavirus: where we are and what we know Cheng ZJ , Zhan ZQ , Xue MS , et al. , 2023 . Public health measures and the control of COVID-19 in China . Clin Rev Allergy Immunol , 64 ( 1 ): 1 - 16 . https://doi.org/10.1007/s12016-021-08900-2 https://doi.org/10.1007/s12016-021-08900-2 Public health measures and the control of COVID-19 in China Conte C , 2021 . Possible link between SARS-CoV-2 infection and Parkinson’s disease: the role of Toll-like receptor 4 . Int J Mol Sci , 22 ( 13 ): 7135 . https://doi.org/10.3390/ijms22137135 https://doi.org/10.3390/ijms22137135 Possible link between SARS-CoV-2 infection and Parkinson’s disease: the role of Toll-like receptor 4 Corman VM , Landt O , Kaiser M , et al. , 2020 . Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR . Euro Surveill , 25 ( 3 ): 2000045 . https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045 https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045 Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Corman VM , Haage VC , Bleicker T , et al. , 2021 . Comparison of seven commercial SARS-CoV-2 rapid point-of-care antigen tests: a single-centre laboratory evaluation study . Lancet Microbe , 2 ( 7 ): e311 - e319 . https://doi.org/10.1016/S2666-5247(21)00056-2 https://doi.org/10.1016/S2666-5247(21)00056-2 Comparison of seven commercial SARS-CoV-2 rapid point-of-care antigen tests: a single-centre laboratory evaluation study da Silva SJR , da Silva CTA , Guarines KM , et al. , 2020 . Clinical and laboratory diagnosis of SARS-CoV-2, the virus causing COVID-19 . ACS Infect Dis , 6 ( 9 ): 2319 - 2336 . https://doi.org/10.1021/acsinfecdis.0c00274 https://doi.org/10.1021/acsinfecdis.0c00274 Clinical and laboratory diagnosis of SARS-CoV-2, the virus causing COVID-19 Dai WH , Zhang B , Jiang XM , et al. , 2020 . Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease . Science , 368 ( 6497 ): 1331 - 1335 . https://doi.org/10.1126/science.abb4489 https://doi.org/10.1126/science.abb4489 Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease Dheda K , Ruhwald M , Theron G , et al. , 2013 . Point-of-care diagnosis of tuberculosis: past, present and future . Respirology , 18 ( 2 ): 217 - 232 . https://doi.org/10.1111/resp.12022 https://doi.org/10.1111/resp.12022 Point-of-care diagnosis of tuberculosis: past, present and future Dhochak N , Singhal T , Kabra SK , et al. , 2020 . Pathophysiology of COVID-19: why children fare better than adults? Indian J Pediatr , 87 ( 7 ): 537 - 546 . https://doi.org/10.1007/s12098-020-03322-y https://doi.org/10.1007/s12098-020-03322-y Pathophysiology of COVID-19: why children fare better than adults? Dou YZ , Su J , Chen SX , et al. , 2022 . A smartphone-based three-in-one biosensor for co-detection of SARS-CoV-2 viral RNA, antigen and antibody . Chem Commun , 58 ( 41 ): 6108 - 6111 . https://doi.org/10.1039/D2CC01297A https://doi.org/10.1039/D2CC01297A A smartphone-based three-in-one biosensor for co-detection of SARS-CoV-2 viral RNA, antigen and antibody Fang YC , Zhang HQ , Xie JC , et al. , 2020 . Sensitivity of chest CT for COVID-19: comparison to RT-PCR . Radiology , 296 ( 2 ): E115 - E117 . https://doi.org/10.1148/radiol.2020200432 https://doi.org/10.1148/radiol.2020200432 Sensitivity of chest CT for COVID-19: comparison to RT-PCR Fang ZF , Sun BQ , Zhu AR , et al. , 2021 . Multiplexed analysis of circulating IgA antibodies for SARS-CoV-2 and common respiratory pathogens in COVID-19 patients . J Med Virol , 93 ( 5 ): 3257 - 3260 . https://doi.org/10.1002/jmv.26829 https://doi.org/10.1002/jmv.26829 Multiplexed analysis of circulating IgA antibodies for SARS-CoV-2 and common respiratory pathogens in COVID-19 patients Fitzpatrick MC , Pandey A , Wells CR , et al. , 2021 . Buyer beware: inflated claims of sensitivity for rapid COVID-19 tests . Lancet , 397 ( 10268 ): 24 - 25 . https://doi.org/10.1016/S0140-6736(20)32635-0 https://doi.org/10.1016/S0140-6736(20)32635-0 Buyer beware: inflated claims of sensitivity for rapid COVID-19 tests Frank MG , Nguyen KH , Ball JB , et al. , 2022 . SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: evidence of PAMP-like properties . Brain Behav Immun , 100 : 267 - 277 . https://doi.org/10.1016/j.bbi.2021.12.007 https://doi.org/10.1016/j.bbi.2021.12.007 SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: evidence of PAMP-like properties Gao JW , Wang CH , Chu YJ , et al. , 2022 . Graphene oxide-graphene Van der Waals heterostructure transistor biosensor for SARS-CoV-2 protein detection . Talanta , 240 : 123197 . https://doi.org/10.1016/j.talanta.2021.123197 https://doi.org/10.1016/j.talanta.2021.123197 Graphene oxide-graphene Van der Waals heterostructure transistor biosensor for SARS-CoV-2 protein detection Gao W , Tian JJ , Huang KL , et al. , 2019 . Ultrafast, universal and visual screening of dual genetically modified elements based on dual super PCR and a lateral flow biosensor . Food Chem , 279 : 246 - 251 . https://doi.org/10.1016/j.foodchem.2018.12.013 https://doi.org/10.1016/j.foodchem.2018.12.013 Ultrafast, universal and visual screening of dual genetically modified elements based on dual super PCR and a lateral flow biosensor Gao YP , Huang KJ , Wang FT , et al. , 2022 . Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications . Analyst , 147 ( 15 ): 3396 - 3414 . https://doi.org/10.1039/D2AN00556E https://doi.org/10.1039/D2AN00556E Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications García-Fiñana M , Buchan IE , 2021 . Rapid antigen testing in COVID-19 responses: SARS-CoV-2 transmission was reduced with measures centered on rapid antigen testing . Science , 372 ( 6542 ): 571 - 572 . https://doi.org/10.1126/science.abi6680 https://doi.org/10.1126/science.abi6680 Rapid antigen testing in COVID-19 responses: SARS-CoV-2 transmission was reduced with measures centered on rapid antigen testing Gaugler S , Sottas PE , Blum K , et al , 2021 . Fully automated dried blood spot sample handling and extraction for serological testing of SARS-CoV-2 antibodies . Drug Test Anal , 13 ( 1 ): 223 - 226 . https://doi.org/10.1002/dta.2946 https://doi.org/10.1002/dta.2946 Fully automated dried blood spot sample handling and extraction for serological testing of SARS-CoV-2 antibodies Gili A , Paggi R , Russo C , et al. , 2021 . Evaluation of Lumipulse® G SARS-CoV-2 antigen assay automated test for detecting SARS-CoV-2 nucleocapsid protein (NP) in nasopharyngeal swabs for community and population screening . Int J Infect Dis , 105 : 391 - 396 . https://doi.org/10.1016/j.ijid.2021.02.098 https://doi.org/10.1016/j.ijid.2021.02.098 Evaluation of Lumipulse® G SARS-CoV-2 antigen assay automated test for detecting SARS-CoV-2 nucleocapsid protein (NP) in nasopharyngeal swabs for community and population screening Gill JL , Williams JW , Jackson ST , et al. , 2009 . Pleistocene megafaunal collapse, novel plant communities, and enhanced fire regimes in North America . Science , 326 ( 5956 ): 1100 - 1103 . https://doi.org/10.1126/science.1179504 https://doi.org/10.1126/science.1179504 Pleistocene megafaunal collapse, novel plant communities, and enhanced fire regimes in North America Greenwood D , Richard S , Barer M , et al. , 2012 . Medical Microbiology . Elsevier , Amsterdam , The Netherlands . Medical Microbiology Grubaugh ND , Ladner JT , Lemey P , et al. , 2019 . Tracking virus outbreaks in the twenty-first century . Nat Microbiol , 4 ( 1 ): 10 - 19 . https://doi.org/10.1038/s41564-018-0296-2 https://doi.org/10.1038/s41564-018-0296-2 Tracking virus outbreaks in the twenty-first century Gu W , Miller S , Chiu CY , 2019 . Clinical metagenomic next-generation sequencing for pathogen detection . Annu Rev Pathol , 14 : 319 - 338 . https://doi.org/10.1146/annurev-pathmechdis-012418-012751 https://doi.org/10.1146/annurev-pathmechdis-012418-012751 Clinical metagenomic next-generation sequencing for pathogen detection Guo M , Tao WY , Flavell RA , et al. , 2021 . Potential intestinal infection and faecal-oral transmission of SARS-CoV-2 . Nat Rev Gastroenterol Hepatol , 18 ( 4 ): 269 - 283 . https://doi.org/10.1038/s41575-021-00416-6 https://doi.org/10.1038/s41575-021-00416-6 Potential intestinal infection and faecal-oral transmission of SARS-CoV-2 Harcourt J , Tamin A , Lu XY , et al. , 2020 . Severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States . Emerg Infect Dis , 26 ( 6 ): 1266 - 1273 . https://doi.org/10.3201/eid2606.200516 https://doi.org/10.3201/eid2606.200516 Severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States Hirotsu Y , Maejima M , Shibusawa M , et al. , 2020 . Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients . Int J Infect Dis , 99 : 397 - 402 . https://doi.org/10.1016/j.ijid.2020.08.029 https://doi.org/10.1016/j.ijid.2020.08.029 Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients Hirotsu Y , Maejima M , Shibusawa M , et al. , 2021a . Prospective study of 1308 nasopharyngeal swabs from 1033 patients using the LUMIPULSE SARS-CoV-2 antigen test: comparison with RT-qPCR . Int J Infect Dis , 105 : 7 - 14 . https://doi.org/10.1016/j.ijid.2021.02.005 https://doi.org/10.1016/j.ijid.2021.02.005 Prospective study of 1308 nasopharyngeal swabs from 1033 patients using the LUMIPULSE SARS-CoV-2 antigen test: comparison with RT-qPCR Hirotsu Y , Sugiura H , Maejima M , et al. , 2021b . Comparison of Roche and Lumipulse quantitative SARS-CoV-2 antigen test performance using automated systems for the diagnosis of COVID-19 . Int J Infect Dis , 108 : 263 - 269 . https://doi.org/10.1016/j.ijid.2021.05.067 https://doi.org/10.1016/j.ijid.2021.05.067 Comparison of Roche and Lumipulse quantitative SARS-CoV-2 antigen test performance using automated systems for the diagnosis of COVID-19 Holland LA , Kaelin EA , Maqsood R , et al. , 2020 . An 81-nucleotide deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona (January to March 2020) . J Virol , 94 ( 14 ): e00711-20 . https://doi.org/10.1128/JVI.00711-20 https://doi.org/10.1128/JVI.00711-20 An 81-nucleotide deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona (January to March 2020) Hu XJ , Deng QY , Li JM , et al. , 2020 . Development and clinical application of a rapid and sensitive loop-mediated isothermal amplification test for SARS-CoV-2 infection . mSphere , 5 ( 4 ): e00808-20 . https://doi.org/10.1128/mSphere.00808-20 https://doi.org/10.1128/mSphere.00808-20 Development and clinical application of a rapid and sensitive loop-mediated isothermal amplification test for SARS-CoV-2 infection Huang L , Tian SL , Zhao WH , et al. , 2020 . Multiplexed detection of biomarkers in lateral-flow immunoassays . Analyst , 145 ( 8 ): 2828 - 2840 . https://doi.org/10.1039/C9AN02485A https://doi.org/10.1039/C9AN02485A Multiplexed detection of biomarkers in lateral-flow immunoassays Iscove NN , Barbara M , Gu M , et al. , 2002 . Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA . Nat Biotechnol , 20 ( 9 ): 940 - 943 . https://doi.org/10.1038/nbt729 https://doi.org/10.1038/nbt729 Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA Islam A , Sangkham S , Tiwari A , et al. , 2022a . Association between global monkeypox cases and meteorological factors . Int J Environ Res Public Health , 19 ( 23 ): 15638 . https://doi.org/10.3390/ijerph192315638 https://doi.org/10.3390/ijerph192315638 Association between global monkeypox cases and meteorological factors Islam A , Hasan MN , Ahammed T , et al. , 2022b . Association of household fuel with acute respiratory infection (ARI) under-five years children in Bangladesh . Front Public Health , 10 : 985445 . https://doi.org/10.3389/fpubh.2022.985445 https://doi.org/10.3389/fpubh.2022.985445 Association of household fuel with acute respiratory infection (ARI) under-five years children in Bangladesh Islam A , Hemo MK , Chopra H , et al. , 2022c . Old enemy with a new face: re-emerging monkeypox disease-an update . J Pure Appl Microbiol , 16 ( S1 ): 2972 - 2988 . https://doi.org/10.22207/JPAM.16.SPL1.18 https://doi.org/10.22207/JPAM.16.SPL1.18 Old enemy with a new face: re-emerging monkeypox disease-an update Islam A , Haque A , Rahman A , et al. , 2022d . A review on measures to rejuvenate immune system: natural mode of protection against coronavirus infection . Front Immunol , 13 : 837290 . https://doi.org/10.3389/fimmu.2022.837290 https://doi.org/10.3389/fimmu.2022.837290 A review on measures to rejuvenate immune system: natural mode of protection against coronavirus infection Islam A , Ahammed T , Noor STA , 2022e . An estimation of five-decade long monkeypox case fatality rate: systematic review and meta-analysis . J Pure Appl Microbiol , 16 ( S1 ): 3036 - 3047 . https://doi.org/10.22207/JPAM.16.SPL1.16 https://doi.org/10.22207/JPAM.16.SPL1.16 An estimation of five-decade long monkeypox case fatality rate: systematic review and meta-analysis Islam A , Rahman A , Jakariya M , et al. , 2023a . A 30-day follow-up study on the prevalence of SARS-CoV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity . Sci Total Environ , 858 : 159350 . https://doi.org/10.1016/j.scitotenv.2022.159350 https://doi.org/10.1016/j.scitotenv.2022.159350 A 30-day follow-up study on the prevalence of SARS-CoV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity Islam A , Adeiza SS , Amin R , et al. , 2023b . A bibliometric study on Marburg virus research with prevention and control strategies . Front Trop Dis , 3 : 1068364 . https://doi.org/10.3389/fitd.2022.1068364 https://doi.org/10.3389/fitd.2022.1068364 A bibliometric study on Marburg virus research with prevention and control strategies Islam A , Hossen F , Rahman A , et al. , 2023c . An opinion on Wastewater-Based Epidemiological Monitoring (WBEM) with Clinical Diagnostic Test (CDT) for detecting high-prevalence areas of community COVID-19 infections . Curr Opin Environ Sci Health , 31 : 100396 . https://doi.org/10.1016/j.coesh.2022.100396 https://doi.org/10.1016/j.coesh.2022.100396 An opinion on Wastewater-Based Epidemiological Monitoring (WBEM) with Clinical Diagnostic Test (CDT) for detecting high-prevalence areas of community COVID-19 infections Jakariya M , Ahmed F , Islam A , et al. , 2022 . Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities . Environ Pollut , 311 : 119679 . https://doi.org/10.1016/j.envpol.2022.119679 https://doi.org/10.1016/j.envpol.2022.119679 Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities Kames J , Holcomb DD , Kimchi O , et al. , 2020 . Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design . Sci Rep , 10 : 15643 . https://doi.org/10.1038/s41598-020-72533-2 https://doi.org/10.1038/s41598-020-72533-2 Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design Karp DG , Danh K , Espinoza NF , et al. , 2020 . A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots . Sci Rep , 10 : 20188 . https://doi.org/10.1038/s41598-020-76913-6 https://doi.org/10.1038/s41598-020-76913-6 A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots Kobayashi Y , Mitsudomi T , 2013 . Management of ground-glass opacities: should all pulmonary lesions with ground-glass opacity be surgically resected? Transl Lung Cancer Res , 2 ( 5 ): 354 - 363 . https://doi.org/10.3978/j.issn.2218-6751.2013.09.03 https://doi.org/10.3978/j.issn.2218-6751.2013.09.03 Management of ground-glass opacities: should all pulmonary lesions with ground-glass opacity be surgically resected? Koczula KM , Gallotta A , 2016 . Lateral flow assays . Essays Biochem , 60 ( 1 ): 111 - 120 . https://doi.org/10.1042/EBC20150012 https://doi.org/10.1042/EBC20150012 Lateral flow assays Krüttgen A , Cornelissen CG , Dreher M , et al. , 2021 . Comparison of the SARS-CoV-2 rapid antigen test to the real star SARS-CoV-2 RT PCR kit . J Virol Methods , 288 : 114024 . https://doi.org/10.1016/j.jviromet.2020.114024 https://doi.org/10.1016/j.jviromet.2020.114024 Comparison of the SARS-CoV-2 rapid antigen test to the real star SARS-CoV-2 RT PCR kit Kubina R , Dziedzic A , 2020 . Molecular and serological tests for COVID-19. A comparative review of SARS-CoV-2 coronavirus laboratory and point-of-care diagnostics . Diagnostics , 10 ( 6 ): 434 . https://doi.org/10.3390/diagnostics10060434 https://doi.org/10.3390/diagnostics10060434 Molecular and serological tests for COVID-19. A comparative review of SARS-CoV-2 coronavirus laboratory and point-of-care diagnostics Kucirka LM , Lauer SA , Laeyendecker O , et al. , 2020 . Variation in false-negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure . Ann Intern Med , 173 ( 4 ): 262 - 267 . https://doi.org/10.7326/M20-1495 https://doi.org/10.7326/M20-1495 Variation in false-negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure Kuntip N , Japrung D , Pongprayoon P , 2021 . What happens when a complementary DNA meets miR-29a cancer biomarker in complex with a graphene quantum dot . ACS Appl Bio Mater , 4 ( 12 ): 8368 - 8376 . https://doi.org/10.1021/acsabm.1c00943 https://doi.org/10.1021/acsabm.1c00943 What happens when a complementary DNA meets miR-29a cancer biomarker in complex with a graphene quantum dot Kurhade C , Zou J , Xia HJ , et al. , 2023 . Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1 and XBB.1 by parental mRNA vaccine or a BA.5 bivalent booster . Nat Med , 29 ( 2 ): 344 - 347 . https://doi.org/10.1038/s41591-022-02162-x https://doi.org/10.1038/s41591-022-02162-x Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1 and XBB.1 by parental mRNA vaccine or a BA.5 bivalent booster la Marca A , Capuzzo M , Paglia T , et al. , 2020 . Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays . Reprod Biomed Online , 41 ( 3 ): 483 - 499 . https://doi.org/10.1016/j.rbmo.2020.06.001 https://doi.org/10.1016/j.rbmo.2020.06.001 Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays Lanciotti RS , Calisher CH , Gubler DJ , et al. , 1992 . Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction . J Clin Microbiol , 30 ( 3 ): 545 - 551 . https://doi.org/10.1128/jcm.30.3.545-551.1992 https://doi.org/10.1128/jcm.30.3.545-551.1992 Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction Leixner G , Voill-Glaninger A , Bonner E , et al. , 2021 . Evaluation of the AMP SARS-CoV-2 rapid antigen test in a hospital setting . Int J Infect Dis , 108 : 353 - 356 . https://doi.org/10.1016/j.ijid.2021.05.063 https://doi.org/10.1016/j.ijid.2021.05.063 Evaluation of the AMP SARS-CoV-2 rapid antigen test in a hospital setting Li F , Ye QH , Chen MT , et al. , 2021 . Cas12aFDet: a CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c . Anal Chim Acta , 1151 : 338248 . https://doi.org/10.1016/j.aca.2021.338248 https://doi.org/10.1016/j.aca.2021.338248 Cas12aFDet: a CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c Li TW , Shao YF , Fu LY , et al. , 2018 . Plasma circular RNA profiling of patients with gastric cancer and their droplet digital RT-PCR detection . J Mol Med , 96 ( 1 ): 85 - 96 . https://doi.org/10.1007/s00109-017-1600-y https://doi.org/10.1007/s00109-017-1600-y Plasma circular RNA profiling of patients with gastric cancer and their droplet digital RT-PCR detection Li YY , Yang X , Zhao WA , 2017 . Emerging microtechnologies and automated systems for rapid bacterial identification and antibiotic susceptibility testing . SLAS Technol , 22 ( 6 ): 585 - 608 . https://doi.org/10.1177/2472630317727519 https://doi.org/10.1177/2472630317727519 Emerging microtechnologies and automated systems for rapid bacterial identification and antibiotic susceptibility testing Li ZT , Yi YX , Luo XM , et al. , 2020 . Development and clin ical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J Med Virol , 92 ( 9 ): 1518 - 1524 . https://doi.org/10.1002/jmv.25727 https://doi.org/10.1002/jmv.25727 Development and clin Liu J , Gratz J , Amour C , et al. , 2016 . Optimization of quantitative PCR methods for enteropathogen detection . PLoS ONE , 11 ( 6 ): e0158199 . https://doi.org/10.1371/journal.pone.0158199 https://doi.org/10.1371/journal.pone.0158199 Optimization of quantitative PCR methods for enteropathogen detection Lv DF , Ying QM , Weng YS , et al. , 2020 . Dynamic change process of target genes by RT-PCR testing of SARS-CoV-2 during the course of a Coronavirus Disease 2019 patient . Clin Chim Acta , 506 : 172 - 175 . https://doi.org/10.1016/j.cca.2020.03.032 https://doi.org/10.1016/j.cca.2020.03.032 Dynamic change process of target genes by RT-PCR testing of SARS-CoV-2 during the course of a Coronavirus Disease 2019 patient Mackay IM , 2004 . Real-time PCR in the microbiology laboratory . Clin Microbiol Infect , 10 ( 3 ): 190 - 212 . https://doi.org/10.1111/j.1198-743X.2004.00722.x https://doi.org/10.1111/j.1198-743X.2004.00722.x Real-time PCR in the microbiology laboratory Mahalakshmi AM , Ray B , Tuladhar S , et al. , 2021 . Does COVID-19 contribute to development of neurological disease? Immun Inflamm Dis , 9 ( 1 ): 48 - 58 . https://doi.org/10.1002/iid3.387 https://doi.org/10.1002/iid3.387 Does COVID-19 contribute to development of neurological disease? Manenti A , Maggetti M , Casa E , et al. , 2020 . Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples . J Med Virol , 92 ( 10 ): 2096 - 2104 . https://doi.org/10.1002/jmv.25986 https://doi.org/10.1002/jmv.25986 Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples McDade TW , McNally EM , Zelikovich AS , et al. , 2020 . High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay . PLoS ONE , 15 ( 8 ): e0237833 . https://doi.org/10.1371/journal.pone.0237833 https://doi.org/10.1371/journal.pone.0237833 High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay Menchinelli G , Bordi L , Liotti FM , et al. , 2021 . Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups . Clin Chem Lab Med , 59 ( 8 ): 1468 - 1476 . https://doi.org/10.1515/cclm-2021-0182 https://doi.org/10.1515/cclm-2021-0182 Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups Mercer TR , Salit M , 2021 . Testing at scale during the COVID-19 pandemic . Nat Rev Genet , 22 ( 7 ): 415 - 426 . https://doi.org/10.1038/s41576-021-00360-w https://doi.org/10.1038/s41576-021-00360-w Testing at scale during the COVID-19 pandemic Metzker ML , 2010 . Sequencing technologies—the next generation . Nat Rev Genet , 11 ( 1 ): 31 - 46 . https://doi.org/10.1038/nrg2626 https://doi.org/10.1038/nrg2626 Sequencing technologies—the next generation Miesse PK , Collier BB , Grant RP , 2022 . Monitoring of SARS-CoV-2 antibodies using dried blood spot for at-home collection . Sci Rep , 12 : 5812 . https://doi.org/10.1038/s41598-022-09699-4 https://doi.org/10.1038/s41598-022-09699-4 Monitoring of SARS-CoV-2 antibodies using dried blood spot for at-home collection Mina MJ , Parker R , Larremore DB , 2020 . Rethinking Covid-19 test sensitivity—a strategy for containment . N Engl J Med , 383 ( 22 ): e120 . https://doi.org/10.1056/NEJMp2025631 https://doi.org/10.1056/NEJMp2025631 Rethinking Covid-19 test sensitivity—a strategy for containment Mohammadi MR , Omidi AH , Sabati H , 2022 . Current trends and new methods of detection of SARS-CoV-2 infection . Cell Mol Biomed Rep , 2 ( 3 ): 138 - 150 . https://doi.org/10.55705/cmbr.2022.345025.1047 https://doi.org/10.55705/cmbr.2022.345025.1047 Current trends and new methods of detection of SARS-CoV-2 infection Montesinos I , Gruson D , Kabamba B , et al. , 2020 . Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies . J Clin Virol , 128 : 104413 . https://doi.org/10.1016/j.jcv.2020.104413 https://doi.org/10.1016/j.jcv.2020.104413 Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies Morley GL , Taylor S , Jossi S , et al. , 2020 . Sensitive detection of SARS-CoV-2-specific antibodies in dried blood spot samples . Emerg Infect Dis , 26 ( 12 ): 2970 - 2973 . https://doi.org/10.3201/eid2612.203309 https://doi.org/10.3201/eid2612.203309 Sensitive detection of SARS-CoV-2-specific antibodies in dried blood spot samples Motayo BO , Oluwasemowo OO , Olusola BA , et al. , 2021 . Evolution and genetic diversity of SARS-CoV-2 in Africa using whole genome sequences . Int J Infect Dis , 103 : 282 - 287 . https://doi.org/10.1016/j.ijid.2020.11.190 https://doi.org/10.1016/j.ijid.2020.11.190 Evolution and genetic diversity of SARS-CoV-2 in Africa using whole genome sequences Muhi S , Tayler N , Hoang T , et al. , 2021 . Multi-site assessment of rapid, point-of-care antigen testing for the diagnosis of SARS-CoV-2 infection in a low-prevalence setting: a validation and implementation study . Lancet Reg Health West Pac , 9 : 100115 . https://doi.org/10.1016/j.lanwpc.2021.100115 https://doi.org/10.1016/j.lanwpc.2021.100115 Multi-site assessment of rapid, point-of-care antigen testing for the diagnosis of SARS-CoV-2 infection in a low-prevalence setting: a validation and implementation study Nicol T , Lefeuvre C , Serri O , et al. , 2020 . Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech) . J Clin Virol , 129 : 104511 . https://doi.org/10.1016/j.jcv.2020.104511 https://doi.org/10.1016/j.jcv.2020.104511 Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech) Nie JH , Li QQ , Wu JJ , et al. , 2020 . Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 . Emerg Microbes Infect , 9 ( 1 ): 680 - 686 . https://doi.org/10.1080/22221751.2020.1743767 https://doi.org/10.1080/22221751.2020.1743767 Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 Oran DP , Topol EJ , 2020 . Prevalence of asymptomatic SARS-CoV-2 infection: a narrative review . Ann Intern Med , 173 ( 5 ): 362 - 367 . https://doi.org/10.7326/M20-3012 https://doi.org/10.7326/M20-3012 Prevalence of asymptomatic SARS-CoV-2 infection: a narrative review Oude Munnink BB , Nieuwenhuijse DF , Stein M , et al. , 2020 . Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands . Nat Med , 26 ( 9 ): 1405 - 1410 . https://doi.org/10.1038/s41591-020-0997-y https://doi.org/10.1038/s41591-020-0997-y Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands Pan F , Ye TH , Sun P , et al. , 2020 . Time course of lung changes at chest CT during recovery from coronavirus disease 2019 (COVID-19) . Radiology , 295 ( 3 ): 715 - 721 . https://doi.org/10.1148/radiol.2020200370 https://doi.org/10.1148/radiol.2020200370 Time course of lung changes at chest CT during recovery from coronavirus disease 2019 (COVID-19) Pang B , Xu JY , Liu YM , et al. , 2020 . Isothermal amplification and ambient visualization in a single tube for the detection of SARS-CoV-2 using loop-mediated amplification and CRISPR technology . Anal Chem , 92 ( 24 ): 16204 - 16212 . https://doi.org/10.1021/acs.analchem.0c04047 https://doi.org/10.1021/acs.analchem.0c04047 Isothermal amplification and ambient visualization in a single tube for the detection of SARS-CoV-2 using loop-mediated amplification and CRISPR technology Peeling RW , Olliaro PL , Boeras DI , et al. , 2021 . Scaling up COVID-19 rapid antigen tests: promises and challenges . Lancet Infect Dis , 21 ( 9 ): e290 - e295 . https://doi.org/10.1016/S1473-3099(21)00048-7 https://doi.org/10.1016/S1473-3099(21)00048-7 Scaling up COVID-19 rapid antigen tests: promises and challenges Peto J , 2020 . Covid-19 mass testing facilities could end the epidemic rapidly . BMJ , 368 : m1163 . https://doi.org/10.1136/bmj.m1163 https://doi.org/10.1136/bmj.m1163 Covid-19 mass testing facilities could end the epidemic rapidly Pumford EA , Lu JK , Spaczai I , et al. , 2020 . Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics . Biosens Bioelectron , 170 : 112674 . https://doi.org/10.1016/j.bios.2020.112674 https://doi.org/10.1016/j.bios.2020.112674 Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics Qiu F , Wang HJ , Zhang ZK , et al. , 2020 . Laboratory testing techniques for SARS-CoV-2 . J Southern Med Univ , 40 ( 2 ): 164 - 167 (in Chinese) . https://doi.org/10.12122/j.issn.1673-4254.2020.02.16 https://doi.org/10.12122/j.issn.1673-4254.2020.02.16 Laboratory testing techniques for SARS-CoV-2 Quail MA , Smith M , Coupland P , et al. , 2012 . A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers . BMC Genomics , 13 : 341 . https://doi.org/10.1186/1471-2164-13-341 https://doi.org/10.1186/1471-2164-13-341 A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers Rajpal S , Lakhyani N , Singh AK , et al. , 2021 . Using handpicked features in conjunction with ResNet-50 for improved detection of COVID-19 from chest X-ray images . Chaos Solitons Fractals , 145 : 110749 . https://doi.org/10.1016/j.chaos.2021.110749 https://doi.org/10.1016/j.chaos.2021.110749 Using handpicked features in conjunction with ResNet-50 for improved detection of COVID-19 from chest X-ray images Rutala WA , Weber DJ , 2017 . Guideline for disinfection and sterilization in healthcare facilities , 2008 . Centers for Disease Control and Prevention . https://stacks.‍cdc.‍gov/view/cdc/47378 https://stacks.‍cdc.‍gov/view/cdc/47378 Centers for Disease Control and Prevention Safiabadi Tali SH , LeBlanc JJ , Sadiq Z , et al. , 2021 . Tools and techniques for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 detection . Clin Microbiol Rev , 34 ( 3 ): e00228-20 . https://doi.org/10.1128/CMR.00228-20 https://doi.org/10.1128/CMR.00228-20 Tools and techniques for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 detection Sakib MH , Nishat AA , Islam MT , et al. , 2021 . Computational screening of 645 antiviral peptides against the receptor-binding domain of the spike protein in SARS-CoV-2 . Comput Biol Med , 136 : 104759 . https://doi.org/10.1016/j.compbiomed.2021.104759 https://doi.org/10.1016/j.compbiomed.2021.104759 Computational screening of 645 antiviral peptides against the receptor-binding domain of the spike protein in SARS-CoV-2 Sandle T , 2013 . Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals: Technology, Validation and Current Regulations . Woodhead Publishing Ltd. , Oxford, UK . Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals: Technology, Validation and Current Regulations Santiago I , 2020 . Trends and innovations in biosensors for COVID-19 mass testing . ChemBioChem , 21 ( 20 ): 2880 - 2889 . https://doi.org/10.1002/cbic.202000250 https://doi.org/10.1002/cbic.202000250 Trends and innovations in biosensors for COVID-19 mass testing Shen B , Zheng Y , Zhang X , et al. , 2020 . Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-CoV-2 IgM/IgG . Am J Transl Res , 12 ( 4 ): 1348 - 1354 . Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-CoV-2 IgM/IgG Shi P , Dong YQ , Yan HC , et al. , 2020 . Impact of temperature on the dynamics of the COVID-19 outbreak in China . Sci Total Environ , 728 : 138890 . https://doi.org/10.1016/j.scitotenv.2020.138890 https://doi.org/10.1016/j.scitotenv.2020.138890 Impact of temperature on the dynamics of the COVID-19 outbreak in China Shi R , Shan C , Duan XM , et al. , 2020 . A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2 . Nature , 584 ( 7819 ): 120 - 124 . https://doi.org/10.1038/s41586-020-2381-y https://doi.org/10.1038/s41586-020-2381-y A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2 Shu YL , McCauley J , 2017 . GISAID: global initiative on sharing all influenza data-from vision to reality . Euro Surveill , 22 ( 13 ): 30494 . https://doi.org/10.2807/1560-7917.ES.2017.22.13.30494 https://doi.org/10.2807/1560-7917.ES.2017.22.13.30494 GISAID: global initiative on sharing all influenza data-from vision to reality Singh J , Sharma S , Nara S , 2015 . Evaluation of gold nanoparticle based lateral flow assays for diagnosis of enterobacteriaceae members in food and water . Food Chem , 170 : 470 - 483 . https://doi.org/10.1016/j.foodchem.2014.08.092 https://doi.org/10.1016/j.foodchem.2014.08.092 Evaluation of gold nanoparticle based lateral flow assays for diagnosis of enterobacteriaceae members in food and water Sirois M , 2014 . Laboratory Procedures for Veterinary Technicians, 6th Ed . Elsevier , St. Louis , USA . Laboratory Procedures for Veterinary Technicians, 6th Ed Smyrlaki I , Ekman M , Lentini A , et al. , 2020 . Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR . Nat Commun , 11 : 4812 . https://doi.org/10.1038/s41467-020-18611-5 https://doi.org/10.1038/s41467-020-18611-5 Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR Soni A , Herbert C , Filippaios A , et al. , 2022 . Comparison of rapid antigen tests’ performance between Delta and Omicron variants of SARS-CoV-2: secondary analysis from a serial home self-testing study . Ann Intern Med , 175 ( 12 ): 1685 - 1692 . https://doi.org/10.7326/M22-0760 https://doi.org/10.7326/M22-0760 Comparison of rapid antigen tests’ performance between Delta and Omicron variants of SARS-CoV-2: secondary analysis from a serial home self-testing study Soto I , Zamorano-Illanes R , Becerra R , et al. , 2023 . A new COVID-19 detection method based on CSK/QAM visible light communication and machine learning . Sensors , 23 ( 3 ): 1533 . https://doi.org/10.3390/s23031533 https://doi.org/10.3390/s23031533 A new COVID-19 detection method based on CSK/QAM visible light communication and machine learning Steinman JB , Lum FM , Ho PPK , et al. , 2020 . Reduced development of COVID-19 in children reveals molecular checkpoints gating pathogenesis illuminating potential therapeutics . Proc Natl Acad Sci USA , 117 ( 40 ): 24620 - 24626 . https://doi.org/10.1073/pnas.2012358117 https://doi.org/10.1073/pnas.2012358117 Reduced development of COVID-19 in children reveals molecular checkpoints gating pathogenesis illuminating potential therapeutics Sun T , Guan J , 2020 . Novel coronavirus and the central nervous system . Eur J Neurol , 27 ( 9 ): e52 . https://doi.org/10.1111/ene.14227 https://doi.org/10.1111/ene.14227 Novel coronavirus and the central nervous system Taleghani N , Taghipour F , 2021 . Diagnosis of COVID-19 for controlling the pandemic: a review of the state-of-the-art . Biosens Bioelectron , 174 : 112830 . https://doi.org/10.1016/j.bios.2020.112830 https://doi.org/10.1016/j.bios.2020.112830 Diagnosis of COVID-19 for controlling the pandemic: a review of the state-of-the-art Tan CW , Chia WN , Qin XJ , et al. , 2020 . A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction . Nat Biotechnol , 38 ( 9 ): 1073 - 1078 . https://doi.org/10.1038/s41587-020-0631-z https://doi.org/10.1038/s41587-020-0631-z A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction Tsang NNY , So HC , Ng KY , et al. , 2021 . Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-PCR testing: a systematic review and meta-analysis . Lancet Infect Dis , 21 ( 9 ): 1233 - 1245 . https://doi.org/10.1016/S1473-3099(21)00146-8 https://doi.org/10.1016/S1473-3099(21)00146-8 Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-PCR testing: a systematic review and meta-analysis Verkhratsky A , Li Q , Melino S , et al. , 2020 . Can COVID-19 pandemic boost the epidemic of neurodegenerative diseases? Biol Direct , 15 : 28 . https://doi.org/10.1186/s13062-020-00282-3 https://doi.org/10.1186/s13062-020-00282-3 Can COVID-19 pandemic boost the epidemic of neurodegenerative diseases? Wang CJ , Li Y , Pan YC , et al. , 2022 . Clinical and immune response characteristics among vaccinated persons infected with SARS-CoV-2 delta variant: a retrospective study . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 11 ): 899 - 914 . https://doi.org/10.1631/jzus.B2200054 https://doi.org/10.1631/jzus.B2200054 Clinical and immune response characteristics among vaccinated persons infected with SARS-CoV-2 delta variant: a retrospective study Wang R , Qian CY , Pang YA , et al. , 2021 . opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-CoV-2 detection . Biosens Bioelectron , 172 : 112766 . https://doi.org/10.1016/j.bios.2020.112766 https://doi.org/10.1016/j.bios.2020.112766 opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-CoV-2 detection Wang XN , Zhu YS , Jiang HW , et al. , 2020 . Detection methods of SARS-CoV-2 . Chem Life , 40 ( 8 ):‍ 1258 - 1269 (in Chinese) . https://doi.org/10.13488/j.smhx.20200280 https://doi.org/10.13488/j.smhx.20200280 Detection methods of SARS-CoV-2 Wang YH , Dong CJ , Hu Y , et al. , 2020 . Temporal changes of CT findings in 90 patients with COVID-19 pneumonia: a longitudinal study . Radiology , 296 ( 2 ): E55 - E64 . https://doi.org/10.1148/radiol.2020200843 https://doi.org/10.1148/radiol.2020200843 Temporal changes of CT findings in 90 patients with COVID-19 pneumonia: a longitudinal study Wang YW , Ma DD , Zhang GP , et al. , 2022 . An electrochemical immunosensor based on SPA and rGO-PEI-Ag-Nf for the detection of arsanilic acid . Molecules , 27 ( 1 ): 172 . https://doi.org/10.3390/molecules27010172 https://doi.org/10.3390/molecules27010172 An electrochemical immunosensor based on SPA and rGO-PEI-Ag-Nf for the detection of arsanilic acid Wen JQ , Cheng YF , Ling RS , et al. , 2020 . Antibody-dependent enhancement of coronavirus . Int J Infect Dis , 100 : 483 - 489 . https://doi.org/10.1016/j.ijid.2020.09.015 https://doi.org/10.1016/j.ijid.2020.09.015 Antibody-dependent enhancement of coronavirus Wu F , Zhao S , Yu B , et al. , 2020 . A new coronavirus associated with human respiratory disease in China . Nature , 579 ( 7798 ): 265 - 269 . https://doi.org/10.1038/s41586-020-2008-3 https://doi.org/10.1038/s41586-020-2008-3 A new coronavirus associated with human respiratory disease in China Wu Q , Wu W , Chen FF , et al. , 2022 . Highly sensitive and selective surface plasmon resonance biosensor for the detection of SARS-CoV-2 spike S1 protein . Analyst , 147 ( 12 ): 2809 - 2818 . Highly sensitive and selective surface plasmon resonance biosensor for the detection of SARS-CoV-2 spike S1 protein Xu Z , Shi L , Wang YJ , et al. , 2020 . Pathological findings of COVID-19 associated with acute respiratory distress syndrome . Lancet Respir Med , 8 ( 4 ): 420 - 422 . https://doi.org/10.1016/S2213-2600(20)30076-X https://doi.org/10.1016/S2213-2600(20)30076-X Pathological findings of COVID-19 associated with acute respiratory distress syndrome Yadav S , Sadique MA , Ranjan P , et al. , 2021 . SERS based lateral flow immunoassay for point-of-care detection of SARS-CoV-2 in clinical samples . ACS Appl Bio Mater , 4 ( 4 ): 2974 - 2995 . https://doi.org/10.1021/acsabm.1c00102 https://doi.org/10.1021/acsabm.1c00102 SERS based lateral flow immunoassay for point-of-care detection of SARS-CoV-2 in clinical samples Yan C , Cui J , Huang L , et al. , 2020 . Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay . Clin Microbiol Infect , 26 ( 6 ): 773 - 779 . https://doi.org/10.1016/j.cmi.2020.04.001 https://doi.org/10.1016/j.cmi.2020.04.001 Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay Yan SJ , Ahmad KZ , Warden AR , et al. , 2021 . One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA . Biosens Bioelectron , 193 : 113535 . https://doi.org/10.1016/j.bios.2021.113535 https://doi.org/10.1016/j.bios.2021.113535 One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA Yang WJ , Cao QQ , Qin L , et al. , 2020 . Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19): a multi-center study in Wenzhou city, Zhejiang, China . Journal of Infection , 80 ( 4 ): 388 - 393 . https://doi.org/10.1016/j.jinf.2020.02.016 https://doi.org/10.1016/j.jinf.2020.02.016 Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19): a multi-center study in Wenzhou city, Zhejiang, China Yuan SJ , Pan Y , Xia Y , et al. , 2021 . Development and validation of an individualized nomogram for early prediction of the duration of SARS-CoV-2 shedding in COVID-19 patients with non-severe disease . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 4 ): 318 - 329 . https://doi.org/10.1631/jzus.B2000608 https://doi.org/10.1631/jzus.B2000608 Development and validation of an individualized nomogram for early prediction of the duration of SARS-CoV-2 shedding in COVID-19 patients with non-severe disease Yüce M , Filiztekin E , Özkaya KG , 2021 . COVID-19 diagnosis—a review of current methods . Biosens Bioelectron , 172 : 112752 . https://doi.org/10.1016/j.bios.2020.112752 https://doi.org/10.1016/j.bios.2020.112752 COVID-19 diagnosis—a review of current methods Yue C , Song WL , Wang L , et al. , 2023 . ACE2 binding and antibody evasion in enhanced transmissibility of XBB.1.5 . Lancet Infect Dis , 23 ( 3 ): 278 - 280 . https://doi.org/10.1016/S1473-3099(23)00010-5 https://doi.org/10.1016/S1473-3099(23)00010-5 ACE2 binding and antibody evasion in enhanced transmissibility of XBB.1.5 Yue L , Xie TH , Yang T , et al. , 2022 . A third booster dose may be necessary to mitigate neutralizing antibody fading after inoculation with two doses of an inactivated SARS-CoV-2 vaccine . J Med Virol , 94 ( 1 ): 35 - 38 . https://doi.org/10.1002/jmv.27334 https://doi.org/10.1002/jmv.27334 A third booster dose may be necessary to mitigate neutralizing antibody fading after inoculation with two doses of an inactivated SARS-CoV-2 vaccine Zava TT , Zava DT , 2021 . Validation of dried blood spot sample modifications to two commercially available COVID-19 IgG antibody immunoassays . Bioanalysis , 13 ( 1 ): 13 - 28 . https://doi.org/10.4155/bio-2020-0289 https://doi.org/10.4155/bio-2020-0289 Validation of dried blood spot sample modifications to two commercially available COVID-19 IgG antibody immunoassays Zhang CY , Shi DM , Li XY , et al. , 2022 . Microfluidic electrochemical magnetoimmunosensor for ultrasensitive detection of interleukin-6 based on hybrid of AuNPs and graphene . Talanta , 240 : 123173 . https://doi.org/10.1016/j.talanta.2021.123173 https://doi.org/10.1016/j.talanta.2021.123173 Microfluidic electrochemical magnetoimmunosensor for ultrasensitive detection of interleukin-6 based on hybrid of AuNPs and graphene Zhang YM , Zhang Y , Xie KB , 2020 . Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice . Mol Breed , 40 : 11 . https://doi.org/10.1007/s11032-019-1092-2 https://doi.org/10.1007/s11032-019-1092-2 Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice Zhu N , Zhang DY , Wang WL , et al. , 2020 . A novel coronavirus from patients with pneumonia in China, 2019 . N Engl J Med , 382 ( 8 ): 727 - 733 . https://doi.org/10.1056/NEJMoa2001017 https://doi.org/10.1056/NEJMoa2001017 A novel coronavirus from patients with pneumonia in China, 2019 Writing: Mingtao LIU, Jiali LYU, Xianhui ZHENG, and Zhiman LIANG. Research and paragraph contributions: Baoying LEI, Huihuang CHEN, and Yiyin MAI. Figure: Zhiman LIANG. Conceptualization and supervision: Huimin HUANG and Baoqing SUN. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Emergency Key Project of Guangzhou Laboratory (No. EKPG21-30-2), China. 10.1631/jzus.B2200625 1673-1581（2023）06-0463-22 刘铭涛,吕佳丽,郑贤惠等.中国视角下的新冠肺炎最新诊断方法演进[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(06):463-484. 刘铭涛,吕佳丽,郑贤惠等.中国视角下的新冠肺炎最新诊断方法演进[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(06):463-484. This work was supported by the Emergency Key Project of Guangzhou Laboratory (No. EKPG21-30-2), China. 2022-12-11 2023-02-27 2023-06-15 2023-07-04 2023-05-16 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 38648178 Mingtao LIU, Jiali LYU, Xianhui ZHENG, Zhiman LIANG, Baoying LEI, Huihuang CHEN, Yiyin MAI, Huimin HUANG, and Baoqing SUN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 6 24