key: cord-1045394-pobo8rsx
authors: Lu, Yanjun; Sun, Ziyong
title: SARS-CoV-2 molecular diagnostics in China
date: 2022-03-04
journal: Clin Lab Med
DOI: 10.1016/j.cll.2022.03.003
sha: 7d41b408d4ff2b86aad471cea59deb8400dbdf82
doc_id: 1045394
cord_uid: pobo8rsx

The COVID-19 pandemic remains a significant problem involving health systems worldwide. Accurate and early detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is critical for minimizing spread and initiating treatment. Several diagnostic methods are reported for detecting the coronavirus in clinical, research, and public health laboratories. Real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) is considered the gold standard, however, due to require skilled personnel and special equipment, rapid antigen tests have been developed and used as first line screening. The serological testing of antibodies can also be used to enhance the detection sensitivity and accuracy which are used to assess the overall infection rate in the community. This review will summarize the molecular techniques and serological 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.

Since the outbreak of COVID-19, the number of infected people has been increasing rapidly worldwide 1, 2 . As of Feb 22, 2022 , more than 420 million confirmed cases of COVID-19 and over 5.8 million deaths worldwide had been reported 3 . 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 4 . One of the successful strategies in China is rapid and extensive detection of 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 29903 nt (GenBank, MN908947.3) in length 5, 6 . 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 6, 7 . 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 8, 9 , while only shared about 79% and 50% sequences with SARS-CoV and MERS-CoV 10 .

According to Diagnosis & Treatment Scheme for Coronavirus Disease 2019 (7th Edition) in China, three 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 11 . Numerous commercial kits for SARS-CoV-2 have been developed and utilized 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 nucleic acids (24 kits), antibodies (25 kits) and antigens (2 kits) 12 . High-throughput sequencing, RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP) have been widely used for SARS-CoV-2 nucleic acid detection [13] [14] [15] , and RT-PCR is recommended as a guideline for the COVID-19 diagnosis and treatment program in China 16 . The serological 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 serological 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. are designed to target various RNA sequences within six genes of SARS-CoV-2 including ORF1a/b, ORF1b-nsp14 (50-UTR), RdRp (RNAdependent RNA polymerase), S, E, N1/N2/N3 and RdRp/Hel (RNA-dependent RNApolymerase/helicase) 17 . 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 18 . The CDC in America recommends three nucleocapsid targets (N1, N2 and N3), while the Europe recommends initial screening with E gene followed by confirmation targeting the RdRp 19, 20 . SARS-CoV-2 has low homology with other batrelated viruses in the ORF1b (involving RdRp), N, and S genes, which are relatively specific genes worth targeting 21, 22 . 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 23 . More recently, Chan et al. 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 24 .

At the start of the epidemic in China, RT-PCR kits were developed rapidly and had the earliest clinical application, however, the accuracy of RT-PCR results was only 30-50% 25 . This is due to 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 25, 26 . Viral loads of respiratory tract specimens are highest in BALF, followed by the 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 sampling easily. Because of the limited sensitivity of RT-PCR, negative result from an oralnasopharyngeal swab was not sufficient for a hospital discharge in China 27, 28 . Actually, inactivation before testing should also be considered to cause false negative results. Thermal inactivation of samples under 56℃ for 30 min was recommended to ensure biosafety for laboratory personnel before SARS-CoV-2 RNA detection 29 . Approximately half of the weakly positive samples were RT-PCR negative after thermal inactivation of SARS-CoV-2 at 56℃ for 45 min in at least one parallel testing 30 .

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 31 . Nucleic acid extraction is one of the most critical steps for nucleic acid detection to ensure the reliability of molecular diagnosis 32 . 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 33 . For each positive sample of external quality assessment (EQA), the percentage agreement of the laboratories using magnetic bead-based extraction method was higher than those using column-based extraction method 33 .

False-negative results could potentially arise from mutations occurring in the primer and probe target regions in the SARSCoV-2 genome 34 . Since RNA viruses have strong 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 SARSCoV-2 infection status 35 .

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 five 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 6 . The first mNGS system related to the ultrahighthroughput 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 36 . 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 37, 38 . However, NGS is currently impractical for routine use in most clinical labs for the diagnosis of SARS-CoV-2 infection due to some limitations, such as the high cost and long testing cycles.

Isothermal amplification of nucleic acid is a method for the rapid and efficient accumulation of nucleic acid at a specific constant temperature. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been introduced to detect SARS-CoV-2 with a series of 4 targetspecific primers targeting 6-different regions of the genome sequences in a combined LAMP and reverse transcription-based methodology 39 . RT-LAMP showed a high degree of specificity (99.5%), sensitivity (91.4%) compared to those of RT-qPCR for identification of SARS-CoV-2 40 . Currently, a 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 41 . Relevant products have been also approved by the NMPA in China as potential POCT method in airports, community clinics and hospitals. However, RT-LAMP assays also have some limitations. This technology is more complicated than RT-PCR and involves multiple pairs of primers, limiting the choice of target sites and resolution or specificity 42 .

The CRISPR/Cas is a gene editing tool-box, a combination of guide RNA (CRISPR RNA or crRNA) and Cas enzyme complex, which is 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 43, 44 . Due to yield rapid read-outs and sensitive results of CRISPR, Which is suitable candidates for simple POCT when coupled to lateral flow readouts.

Serological 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 serological evidence for clinical diagnosis and help confirm the diagnosis of patients with negative nucleic acid tests in clinically suspected patients 45 . 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 J o u r n a l P r e -p r o o f IgG antibodies. Particularly, the S1 subunit was more specific than S2 in detecting the SARS-CoV-2specific antibodies 46 . The seroconversion of specific IgM and IgG antibodies against SARS-CoV-2 mainly turn positive in the second or third week after symptom onset, but IgA and IgM were both detectable at the 5th day (median), while IgG appeared on the 14th day (median) in another study 47 . It has been reported that the IgM reached peak within 15-21 days after infection and slowly began to decline, while IgG peaked during 22-39 days and lasted for a longer time 48 . The IgM and IgG antibodies converted to negative around 36 days and over 50 days respectively 47 .

Currently, immunoassays have been developed for detection of COVID-19 infection in serum, plasma and whole blood. Among these strategies, lateral flow immunoassays (LFIA) based on gold particles, up-converting phosphor, or quantum dot fluorescence, along with chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA), are the most promising approaches. The LFIA is user-friendly, cheap, and easily mass-produced, and the main advantage of LFIA is POCT potential usage. 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 (14-25 days after onset of symptoms) of IgG LIFA was ≥ 92.1% 49 . Another study has reported that the sensitivity of NG-Test® was estimated to be 85% [71.9%-92.3%] and the specificity 98.3% [95.0%-100.0%]) for both IgG and IgM when compared to the ELISA Wantai Immunoassay 50 . Another meta-analysis reported the sensitivity/specificity of CLIA, ELISA and lateral flow immunoassay 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 51 . CLIA platforms are widely used as serological techniques for the quantitative detection of specific antigens or antibodies, which are believed to be promising emerging methods for SARS-Cov-2 antibody detection.

Serological 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, serological testing alone cannot be used alone for diagnosis or exclusion of SARS-Cov-2 infection. Furthermore, it is also not suitable for general population screening 52 . False-positive detection of IgM and IgG antibodies has been described, mainly associated with cut-off values of the kit. A weak positive result near the cut-off value is likely to be a false positive 53 . Another reason caused false-positive results is interfering substances in plasma samples (including interferon, rheumatoid factors (RF) and nonspecific antibodies 54 . Additionally, potential cross-reactivity of SARS-COV-2 antibodies with antibodies generated by other coronaviruses probably also results in false-positive results 55 .

Recently, two 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 56 . With RT-PCR assay as the reference standard, the sensitivity, specificity and percentage agreement of NP antigen testing by the fluorescence immunochromatographic (FIC) 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 57 . While 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 58 .

While the application of mNGS technology played an important role in tackling with the pathogen SARS-COV-2 quickly for COVID-19 in the early stage of the epidemic in Wuhan, the RT-PCR will still be the gold standard for the diagnosis of COVID-19 and play 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 is evolving during transmission, the viruses have displayed a large number of genetic variations, which may cause mismatches between primers, probes, and target sequences, and lead to reduced detection performance and false-negative results. So the 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, which also should be the high-throughput and low-cost differential diagnostic technologies.

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