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SARS-CoV-2 Molecular Diagnostics in China

Published:March 03, 2022DOI:https://doi.org/10.1016/j.cll.2022.03.003

      Keywords

      Key points

      • Primers designed to target various RNA sequences within different genes of SARS-CoV-2 affect the sensitivity.
      • Molecular techniques and serological assays widely used in China have the advantages and disadvantages of these techniques.
      • Immunoassays have been developed for detection of COVID-19 but still as a complementary identification assay.

      Introduction

      Since the outbreak of coronavirus disease 2019 (COVID-19), the number of infected people has been increasing rapidly worldwide.
      • Zhu N.
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      • Wang W.
      • et al.
      A novel coronavirus from patients with pneumonia in China, 2019.
      ,
      • Wang C.
      • Horby P.W.
      • Hayden F.G.
      • et al.
      A novel coronavirus outbreak of global health concern.
      As of February 22, 2022, more than 420 million confirmed cases of COVID-19 and over 5.8 million deaths worldwide had been reported.
      • World Health Organization
      Coronavirus disease (COVID-19) outbreak situation.
      With effective prevention and control strategies, China won a significant early victory against COVID-19, and now mainly focuses on preventing the transmission of imported COVID-19.
      • Li Z.
      • Chen Q.
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      Active case finding with case management: the key to tackling the COVID-19 pandemic.
      One of the successful strategies in China is rapid and extensive detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to decrease the risk of transmission by rapidly enabling isolation and contact tracing.
      SARS-CoV-2 is a positive-sense, single-stranded RNA virus, and the whole viral genome is approximately 29,903 nt (GenBank, MN908947.3) in length.
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      Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
      ,
      • Lu R.
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      Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.
      SARS-CoV-2 consists of at least 12 coding regions, including open reading frames (ORF) 1 ab, S, 3, E, M, 7, 8, 9, 10b, N, 13, and 14.
      • Lu R.
      • Zhao X.
      • Li J.
      • et al.
      Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.
      ,
      • Wu A.
      • Peng Y.
      • Huang B.
      • et al.
      Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China.
      Orf1ab and orf1a genes are located at the 5′-end of the genome, which encode pp1ab and pp1a proteins, respectively. The 3′-end of the genome encodes 4 structural proteins including spike, envelope, membrane, and nucleocapsid proteins, as well as accessory proteins. Genomic sequencing revealed that SARS-CoV-2 was closely related to bat-SL-CoVZC45 and bat-CoV RaTG13 with a similarity of 88% and 96.3%, respectively,
      • Lu J.
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      Genomic epidemiology of SARS-CoV-2 in guangdong province, China.
      ,
      • Paraskevis D.
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      • Magiorkinis G.
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      Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event.
      whereas only shared about 79% and 50% sequences with SARS-CoV and MERS-CoV.
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      • Liu W.
      • Zhang Q.
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      RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak.
      According to Diagnosis & Treatment Scheme for Coronavirus Disease 2019 (7th Edition) in China, 3 methods have been used for the diagnosis of SARS-CoV-2 infection, including detection of positive SARS-CoV-2 nucleic acids by reverse transcription–polymerase chain reaction (RT-PCR), viral gene sequencing to detect known SARS-CoV-2 sequences, and the identification of positive SARS-CoV-2–specific IgM and IgG antibodies in serum.
      • Zhao J.Y.
      • Yan J.Y.
      • Qu J.M.
      Interpretations of "diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7).
      Numerous commercial kits for SARS-CoV-2 have been developed and used in the battle against COVID-19. As of November 20, 2020, a total of 51 approved kits for SARS-CoV-2 had been approved by the National Medical Products Administration of China (NMPA), including 24 that detect nucleic acids (), 25 kits that detect antibodies , and 2 kits targeting antigens .
      • Ruhan A.
      • Wang H.
      • Wang W.
      • et al.
      Summary of the detection kits for SARS-CoV-2 approved by the national medical products administration of China and their application for diagnosis of COVID-19.
      High-throughput sequencing, RT-PCR, RT-loop–mediated isothermal amplification (RT-LAMP) have been widely used for SARS-CoV-2 nucleic acid detection,
      • Waggoner J.J.
      • Stittleburg V.
      • Pond R.
      • et al.
      Triplex real-time RT-PCR for Severe acute respiratory syndrome coronavirus 2.
      • Dao Thi V.L.
      • Herbst K.
      • Boerner K.
      • et al.
      A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.
      • Zenker S.
      • Kock F.
      The coronavirus pandemic - a critical discussion of a tourism research agenda.
      and RT-PCR is recommended in the guideline for the COVID-19 diagnosis and treatment program in China.
      General Office of National Health Commission of the People's Republic of China
      Prevention and control scheme for novel coronavirus pneumonia (version 2) [Internet].
      The serologic assays mainly include lateral flow immunoassay (LFIA), chemiluminescence immunoassay (CLIA), or enzyme-linked immunosorbent assay (ELISA), used to detect antibodies produced by individuals exposed to SARS-CoV-2. Some LFIA-based antigen detection kits have been developed recently.
      This review summarizes the molecular techniques and serologic assays widely used in China and discusses the advantages and disadvantages of these techniques. In brief, it is crucial to select appropriate diagnostic methods or combine different methods and other clinical parameters to confirm the SARS-CoV-2 infection status of individuals.

      Reverse transcription––polymerase chain reaction

      Nucleic acid detection is an important diagnostic tool for the clinical diagnosis, segregation, rehabilitation, and discharge of patients, and was also applied as the “gold standard” for the detection of SARS-CoV-2 infection in the early stage of the epidemic. Currently, numerous primers are designed to target various RNA sequences within 6 genes of SARS-CoV-2 including ORF1a/b, ORF1b-nsp14 (50-UTR), RdRp (RNA-dependent RNA polymerase), S, E, N1/N2/N3, and RdRp/Hel (RNA-dependent RNA polymerase/helicase).
      • Abduljalil J.M.
      Laboratory diagnosis of SARS-CoV-2: available approaches and limitations.
      The Chinese Center for Disease Control and Prevention (CDC) recommends the use of primers and fluorescent probes targeting SARS-CoV-2 ORF1ab and nucleocapsid protein (N) gene regions.
      National Institute for Viral Disease Control and Prevention
      Chinese center for disease control and prevention: novel coronavirus national science and technology resource service system[EB/OL].
      The CDC in America recommends two nucleocapsid targets (N1, N2, ), whereas Europe recommends initial screening with E gene followed by confirmation targeting the RdRp.
      • The Food and Drug Administration
      CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel.
      ,
      • Corman V.M.
      • Landt O.
      • Kaiser M.
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      Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
      SARS-CoV-2 has low homology with other bat-related viruses in the ORF1b (involving RdRp), N, and S genes, which are relatively specific genes worth targeting.
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      • Chu D.K.W.
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      • Cheng S.M.S.
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      Recent clinical evaluations have further demonstrated that the N1, N2, and E gene detection assays have better performance than the RdRP and N3 detection assays.
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      • Casto A.M.
      • Huang M.W.
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      Comparative performance of SARS-CoV-2 detection assays using seven different primer-probe sets and one assay kit.
      More recently, Chan and colleagues designed novel primers and probes for real-time RT-PCR detection of RdRp/Helicase (Hel), S and N genes, which was more sensitive than assays targeting other genes.
      • Chan J.F.
      • Yip C.C.
      • To K.K.
      • et al.
      Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR Assay validated in vitro and with clinical specimens.
      At the start of the epidemic in China, RT-PCR kits were developed rapidly and had the earliest clinical application; however, the sensitivity of RT-PCR results was only 30% to 50%.
      • Li N.
      • Wang P.
      • Wang X.
      • et al.
      Molecular diagnosis of COVID-19: current situation and trend in China (Review).
      This is due to a variety of factors, including low viral loads in specimens such as throat swabs and other respiratory samples, samples not being properly preserved, and the technology itself, which would be affected by virus mutation and PCR inhibitor.
      • Li N.
      • Wang P.
      • Wang X.
      • et al.
      Molecular diagnosis of COVID-19: current situation and trend in China (Review).
      ,
      • Ji T.
      • Liu Z.
      • Wang G.
      • et al.
      Detection of COVID-19: a review of the current literature and future perspectives.
      Viral loads of respiratory tract specimens are highest in bronchoalveolar lavage fluid(BALF), followed by sputum, nasal swabs, and pharyngeal swabs; however, in clinical application, nasopharyngeal and oropharyngeal swabs served as the main sample types for clinical testing due to ease of sampling . Because of the limited sensitivity of RT-PCR, a negative results from an oral-nasopharyngeal swab was not sufficient for hospital discharge in China.
      • Wang W.
      • Xu Y.
      • Gao R.
      • et al.
      Detection of SARS-CoV-2 in different types of clinical specimens.
      ,
      • Wang X.
      • Tan L.
      • Wang X.
      • et al.
      Comparison of nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 detection in 353 patients received tests with both specimens simultaneously.
      Virus inactivation before testing should also be considered to cause false-negative results. Thermal inactivation of samples at 56°C for 30 min was recommended to ensure biosafety for laboratory personnel before SARS-CoV-2 RNA detection.
      National Health Commission of the People’s Republic of China
      Technical guide for prevention and control of coronavirus disease 2019 in medical institutions.
      However, approximately half of the weakly positive samples were RT-PCR negative after thermal inactivation of SARS-CoV-2 at 56°C for 45 min in parallel testing.
      • Pan Y.
      • Long L.
      • Zhang D.
      • et al.
      Potential false-negative nucleic acid testing results for severe acute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads.
      A series of assays have been approved by the NMPA with Emergency Use Authorization in response to COVID-19 infection; however, the analytical performance claimed in the corresponding instructions by manufacturers has not been thoroughly validated. In clinical applications, the differences in nucleic acid extraction methods, RT-PCR processes, personnel, or equipment lead to variations in testing results among different laboratories.
      • Sung H.
      • Han M.G.
      • Yoo C.K.
      • et al.
      Nationwide external quality assessment of SARS-CoV-2 molecular testing, South Korea.
      Nucleic acid extraction is one of the most critical steps for nucleic acid detection to ensure the reliability of molecular diagnosis.
      • Ali N.
      • Rampazzo R.C.P.
      • Costa A.D.T.
      • et al.
      Current nucleic acid extraction methods and their implications to point-of-care diagnostics.
      In China, various nucleic acid extraction methods were applied by the laboratories. Among these methods, manual column-based, manual magnetic bead-based, automated column-based, and automated magnetic bead-based methods accounted for 21.3% (198/931), 15.3% (142/931), 1.5% (14/931), and 51.7% (481/931), respectively.
      • Wang Z.
      • Chen Y.
      • Yang J.
      • et al.
      External quality assessment for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical laboratories.
      For each positive sample of external quality assessment, the percentage agreement of the laboratories using magnetic bead-based extraction method was higher than those using column-based extraction method.
      • Wang Z.
      • Chen Y.
      • Yang J.
      • et al.
      External quality assessment for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical laboratories.
      False-negative results could potentially arise from mutations occurring in the primer and probe-target regions in the SARS-CoV-2 genome.
      • Tahamtan A.
      • Ardebili A.
      Real-time RT-PCR in COVID-19 detection: issues affecting the results.
      As RNA viruses have a high degree of genetic variability, mismatches between primers and target sequences caused by mutations can lead to poor detection performance. The results should be validated with different primer sets against the same gene and combined with patient history and other clinical data to accurately determine SARS-CoV-2 infection status.
      • Shen Z.
      • Xiao Y.
      • Kang L.
      • et al.
      Genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease 2019.

      High-throughput sequencing

      In the early stage of the epidemic, the metagenomics next-generation sequencing (mNGS) was used to identify and analyze the genome of SARS-CoV-2 within 5 days by the Chinese CDC. The phylogenetic analysis of these genomes showed that the similarity between the genomic sequence of SARS-CoV-2 and SARS or bat-derived strains were 79% and 88%, respectively.
      • Lu R.
      • Zhao X.
      • Li J.
      • et al.
      Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.
      The first mNGS system related to the ultra-high-throughput sequencer DNBSEQ-T7, with the supporting analysis software and nucleic acid detection kits, has been approved by the NMPA, which can identify and diagnose coronaviruses, including SARS-CoV-2 and other infectious respiratory pathogens, and enable rapid detection of viral sequences.
      China National Medical Products Administration
      Registration information of domestic new coronavirus detection reagents.
      Nanopore sequencing is a third-generation genome sequencing technology providing real-time analysis and rapid insights, which does not require enzymes to amplify samples and directly performs full-length sequencing of SARS-CoV-2 and additional respiratory viruses within a few hours.
      • Li J.
      • Wang H.
      • Mao L.
      • et al.
      Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing.
      ,
      • Wang M.
      • Fu A.
      • Hu B.
      • et al.
      Nanopore targeted sequencing for the accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses.
      However, NGS is currently impractical for routine use in most clinical laboratories for the diagnosis of SARS-CoV-2 infection due to some limitations, such as the high cost and long testing cycles.

      Isothermal Amplification Assays

      Isothermal amplification of nucleic acid is a method for the rapid and efficient accumulation of nucleic acid at a specific constant temperature. RT-LAMP has been introduced to detect SARS-CoV-2 with a series of 4 target-specific primers targeting 6-different regions of the genome in a combined LAMP and reverse transcription-based methodology.
      • Yan C.
      • Cui J.
      • Huang L.
      • et al.
      Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay.
      RT-LAMP showed a high degree of specificity (99.5%), sensitivity (91.4%) compared with RT-qPCR for identification of SARS-CoV-2.
      • Jiang M.
      • Pan W.
      • Arasthfer A.
      • et al.
      Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19.
      Currently, point-of-care testing (POCT) of SARS-CoV-2 in nasal swabs using RT-LAMP from Abbott Diagnostics has been approved by US FDA. However, it is restricted to one sample per run.
      • Carter L.J.
      • Garner L.V.
      • Smoot J.W.
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      Assay techniques and test development for COVID-19 diagnosis.
      Relevant products have been also approved by the NMPA in China as a potential POCT method in airports, community clinics, and hospitals. The limitations of RT-LAMP assays are that the technology is more complicated than RT-PCR and involves multiple pairs of primers, limiting the choice of target sites and resolution or specificity.
      • Li Y.
      • Fan P.
      • Zhou S.
      • et al.
      Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens.

      CRISPR-based newly developed methods

      The CRISPR/Cas is a gene-editing toolbox, a combination of guide RNA (CRISPR RNA or crRNA) and Cas enzyme complex for detecting various target sequences and being applied in diagnostic microbiology and biomedicine. Recently, CRISPR has been developed for the detection of SARS-Cov-2 in China and the clinical sensitivity and specificity are comparable to RT-qPCR.
      • Hou T.
      • Zeng W.
      • Yang M.
      • et al.
      Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.
      ,
      • Qiu F.
      • Wang H.
      • Zhang Z.
      • et al.
      [Laboratory testing techniques for SARS-CoV-2].
      Owing to yield rapid read-outs and sensitive results of CRISPR, which is suitable candidates for simple POCT when coupled with lateral flow readouts.

      Antibody detection assays

      Serologic IgM/IgG antibody detection is suggested as a complementary identification assay to indirectly confirm SARS-CoV-2 infection. Briefly, the detection of specific antibodies can provide serologic evidence for infection and help confirm the diagnosis in patients with negative nucleic acid tests but high clinical suspicion .
      • Jacofsky D.
      • Jacofsky E.M.
      • Jacofsky M.
      Understanding antibody testing for COVID-19.
      In SARS-CoV-2 infection, RBD, S, and N proteins serve as the main antigens to stimulate the immune response of the body, producing IgA, IgM, and IgG antibodies. Particularly, the S1 subunit was more specific than S2 in detecting the SARS-CoV-2-specific antibodies.
      • Okba N.M.A.
      • Muller M.A.
      • Li W.
      • et al.
      Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients.
      The seroconversion of specific IgM and IgG antibodies against SARS-CoV-2 typically turn positive in the second or third week after symptom onset, but IgA and IgM were both detectable at the 5th day (median), whereas IgG appeared on the 14th day (median) in another study.
      • Guo L.
      • Ren L.
      • Yang S.
      • et al.
      Profiling early humoral response to diagnose novel coronavirus disease (COVID-19).
      It has been reported that IgM peaks around 15 to 21 days after infection then slowly began to decline, whereas IgG peaks at 22 to 39 days and lasts for a longer time.
      • Wu L.X.
      • Wang H.
      • Gou D.
      • et al.
      Clinical significance of the serum IgM and IgG to SARS-CoV-2 in coronavirus disease-2019.
      IgM and IgG antibodies convert to negative around 36 days and over 50 days, respectively.
      • Guo L.
      • Ren L.
      • Yang S.
      • et al.
      Profiling early humoral response to diagnose novel coronavirus disease (COVID-19).
      Currently, immunoassays have been developed for the detection of COVID-19 infection in serum, plasma, and whole blood. Among these strategies, LFIAs based on gold particles, up-converting phosphor, or quantum dot fluorescence, along with CLIA and ELISA, are the most promising approaches. The LFIA is user-friendly, cheap, and easily mass-produced, and the main advantage of LFIA is its potential usage for POCT. The diagnostic performance of 7 IgG/IgM LFIA kits has been evaluated for detecting SARS-CoV-2 antibodies in COVID-19 patients, and the specificity was ≥ 90.3% for IgG, ≥ 91.3% for IgM, and ≥97.1% for the combination IgM and IgG. The sensitivity 14 to25 days after onset of symptoms of the IgG LFIA was ≥ 92.1%.
      • Van Elslande J.
      • Houben E.
      • Depypere M.
      • et al.
      Diagnostic performance of seven rapid IgG/IgM antibody tests and the euroimmun IgA/IgG ELISA in COVID-19 patients.
      Another study has reported that the sensitivity of the NG-Test was estimated to be 85% (95% confidence interval [CI]71.9%-92.3%) and the specificity 98.3% (95% CI 95.0%-100.0%) for both IgG and IgM when compared with the ELISA Wantai Immunoassay.
      • Garlantezec R.
      • Heslan C.
      • Tadie E.
      • et al.
      A lateral flow immunoassay test performance in SARS-CoV-2 seroprevalence surveys: a validation study among healthcare workers.
      Another meta-analysis reported the sensitivity/specificity of CLIA, ELISA, and LFIA were 92% (95% CI: 86%-95%)/99% (CI: 97%-99%), 86% (CI: 82%-89%)/99% (CI: 98%-100%), and 78% (CI: 71%-83%)/98% (95% CI: 96%-99%), respectively.
      • Mekonnen D.
      • Mengist H.M.
      • Derbie A.
      • et al.
      Diagnostic accuracy of serological tests and kinetics of severe acute respiratory syndrome coronavirus 2 antibody: a systematic review and meta-analysis.
      CLIA platforms are widely used as serologic techniques for the quantitative detection of specific antigens or antibodies, which are believed to be promising emerging methods for SARS-CoV-2 antibody detection.
      Serologic testing also has some limitations. The slow antibody response to SARS-CoV-2 virus indicates that they cannot be helpful in the early stages of infection. Thus, serologic testing alone cannot be used for diagnosis or exclusion of SARS-CoV-2 infection. Furthermore, it is also not suitable for general population screening.
      General Offce of National Health Commission of the People's Republic of China
      Prevention and control scheme for novel coronavirus pneumonia (version 2) [Internet].
      False-positive detection of IgM and IgG antibodies has been described, mainly associated with the manufacturer-determined cut-off values of the kit. A weak positive result near the cut-off value is likely to be a false positive.
      • Latiano A.
      • Tavano F.
      • Panza A.
      • et al.
      False-positive results of SARS-CoV-2 IgM/IgG antibody tests in sera stored before the 2020 pandemic in Italy.
      Another reason for false-positive results is interfering substances in plasma samples including interferon, rheumatoid factors and nonspecific antibodies.
      • Deeks J.J.
      • Dinnes J.
      • Takwoingi Y.
      • et al.
      Antibody tests for identification of current and past infection with SARS-CoV-2.
      In addition, potential cross-reactivity of SARS-CoV-2 antibodies with antibodies generated by other coronaviruses could also results in false-positive results.
      • Udugama B.
      • Kadhiresan P.
      • Kozlowski H.N.
      • et al.
      Diagnosing COVID-19: the disease and tools for detection.

      Antigen-based assays for SARS-CoV-2

      Recently, 2 antigen-based kits for rapid SARS-CoV-2 detection have been approved by the NMPA of China. These antigen-based detection kits were developed based on LFIA using the double antibody sandwich method.
      China National Medical Products Administration
      Registration information of domestic new coronavirus detection reagents.
      With RT-PCR assay as the reference standard, the sensitivity, specificity, and percentage agreement of nucleocapsid protein antigen testing by the fluorescence immunochromatographic assay was 75.6% (95% CI, 69.0–81.3), 100% (95% CI, 91.1–100), and 80.5% (95% CI, 75.1–84.9) respectively, suggesting high specificity and relatively high sensitivity in SARS-CoV-2 diagnosis in the early phase of infection.
      • Diao B.
      • Wen K.
      • Zhang J.
      • et al.
      Accuracy of a nucleocapsid protein antigen rapid test in the diagnosis of SARS-CoV-2 infection.
      Although antigen tests may detect virus early in infection, they may have lower sensitivity compared with nucleic acid amplification tests and may cross-react with other coronaviruses.
      European Centre for disease Prevention and Control
      An overview of the rapid test situation for COVID-19 diagnosis in the EU/EEA.

      Summary

      Although the application of mNGS technology played an important role in detecting the pathogen SARS-CoV-2 in the early stage of the epidemic in Wuhan, RT-PCR is still the gold standard for the diagnosis of COVID-19 and plays an essential role in patient management as well as infection control. POCT molecular testing platforms such as Qiagen’s, BioFire’s Filmarray and Cepheid’s GeneXpert can deliver fast, safe, simple, and accurate molecular detection of pathogens such as COVID-19, which are believed to be promising emerging methods for SARS-CoV-2 detection.
      SARS-CoV-2 has evolved during the past two years, and the viruses have displayed a large number of genetic variations. This may cause mismatches between primers, probes, and target sequences, and lead to reduced detection performance and false-negative results. To prevent this, RT-PCR primer sets should be updated according to the genetic variants in SARS-CoV-2 genomic sequences.
      In the future, novel epidemics or pandemics may be inevitable. There are various types of pneumonia-related pathogens, including SARS-CoV-2, SARS-CoV, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, mycoplasma, and Chlamydia. It is necessary to focus on the development of detection technologies and supporting reagents that can simultaneously rapidly detect dozens of pathogens, while still being the high-throughput and low-cost differential diagnostic technologies.

      Clinics care points

      • RT-PCR is recommended in the guidelines for the COVID-19 diagnosis and treatment program in China due to simple operation, high throughput screening and super sensitivity.
      • Serological IgM/IgG antibody detection is suggested as a complementary identification assay to indirectly confirm SARS-CoV-2 infection due to detect not the pathogen itself and the slow antibody response.

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