key: cord-0803755-zohdn2f1
authors: Zhang, Xiucai; Meng, Hanyan; Liu, Huihui; Ye, Qing
title: Advances in laboratory detection methods and technology application of SARS‐CoV‐2
date: 2021-12-10
journal: J Med Virol
DOI: 10.1002/jmv.27494
sha: 6fbcdfe59dfbc8a97999edf9fae936ce344074cc
doc_id: 803755
cord_uid: zohdn2f1

At present, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is raging worldwide, and the coronavirus disease 2019 outbreak caused by SARS‐CoV‐2 seriously threatens the life and health of all humankind. There is no specific medicine for novel coronavirus yet. So, laboratory diagnoses of novel coronavirus as soon as possible and isolation of the source of infection play a vital role in preventing and controlling the epidemic. Therefore, selecting appropriate detection techniques and methods is particularly important to improve the efficiency of disease diagnosis and treatment and to curb the outbreak of infectious diseases. In this paper, virus nucleic acid, protein, and serum immunology were reviewed to provide a reference for further developing virus detection technology to provide better prevention and treatment strategies and research ideas for clinicians and researchers.

The reverse transcription-PCR (RT-PCR) process of SARS-CoV-2 includes specimen collection, specimen transportation to the laboratory, specimen lysis, virus RNA extraction and purification, RT-PCR amplification, detection, and analysis. 5 The samples were lysed before RT-PCR amplification, and nucleic acids were extracted to remove potential inhibitors that might hinder target amplification. Both lysis/extraction and RT-PCR amplification can be performed by manually processing the instrument or automated operation. The detection rate of RT-PCR is different in patients with different specimens of COVID-19. As shown in Table 1 , the detection rate of bronchoalveolar lavage fluid, sputum, rectal swabs, and nasopharyngeal swabs was higher by RT-PCR. However, the detection rate of the virus in blood and urine samples is meager.

The RT-PCR of SARS-CoV-2 has the characteristics of high sensitivity, strong specificity, rapidity and accuracy, and mature technology, which is widely used in the screening of SARS-CoV-2.

However, it is necessary to strictly control the quality control of sample collection, detection, result interpretation, and so forth. In addition, to avoid false positive-or false-negative results, misdiagnosis or missed diagnosis may occur.

Except for RT-PCR, NAAT research has been launched to develop portable and rapid diagnostic tests for SARS-CoV-2. Isothermal amplification (IAT) replaces the high-temperature melting step in PCR with special enzymes. As it can be carried out under constant temperature, it does not need expensive equipment such as a thermal cycler. The principle of IATs is thermal denaturation or enzymatic denaturation of nucleic acids, followed by the nucleic acid amplification reaction. 12 Isothermal NAAT technology includes transcription-mediated amplification (TMA), nick enzyme-assisted reaction (NEAR), LAMP, reverse transcription-recombinase polymerase amplification (RPA), and repeating CRISPR-Cas-related systems with short palindromes at regular intervals. The following

sections describe examples of IAT and its current and potential applications.

The reaction mechanism of RT-RPA is relatively simple, but the reaction components are relatively complex. Unlike RT-PCR, RT-RPA does not need complex instruments such as thermal cyclers, thus simplifying the detection process. The ease of use of this isothermal technique makes RT-RPA an attractive candidate for molecular testing. RT-RPA technology has been applied to the detection of other RNA viruses, such as the Ebola virus. 13 However, to date, the data used to detect SARS-CoV-2 are not perfect.

Kim et al. used modified RT-RPA to detect SARS-CoV-2 and achieved a sensitivity of approximately four copies/reaction in a 10-minute reaction using lateral flow immunoassay (LFIA) readings.

Their RT-RPA correctly identified 18 artificial samples produced by adding heat-inactivated virus to flocked nasopharyngeal swabs or saliva. 14 Compared with RT-PCR, when tested on 21 nasal swabs, the sensitivity was 80%, and the specificity was 73%, which was relatively low. 28 The authors of this study believed that although the sensitivity and specificity are poor, the analysis is still valuable in some clinical applications. Other studies using RT-LAMP show that compared with LDT based on RT-PCR, the performance of common sample types using RT-LAMP is different. 29 Generally, the range of reaction copies is consistent with some LDT and commercial RT-PCR 

Gene sequencing technology has been successfully applied to identify unknown viruses, conducive to our rapid response to the out- RT-PCR on single flow cells. 35 The coincidence rate of the two methods was very high, and the positive rate of NGS diagnosis increased by 5.7%

(6 cases were negative by PCR, and 21 cases were uncertain by PCR).

This study demonstrated the feasibility of processing 1536 samples in a total of 17 h (11 h for sequencing and 6 h for analysis). 35 In another study, a low-cost NGS method proved to be highly sensitive to SARS-CoV-2, and its sensitivity was equal to or higher than that of some RT-PCR methods. However, only 10 samples (five positive samples and five negative samples) were tested in this study. If the flux is increased to their proposed workflow of 192 samples within 8 h, it is unclear whether high sensitivity will still occur. Compared with limited samples (31 positives and 33 negatives), the coincidence rate between NGS and RT-PCR was 100%. 36 Generally, some data support the potential of NGS as a diagnostic tool for SARS-CoV-2, but further analysis is needed to understand its advantages and limitations.

In summary, NGS is conducive to the initial identification and indepth study of SARS-CoV-2, and its accuracy is very high. However, NGS requires more equipment, takes more time and costs, and is unsuitable for rapid clinical screening. Although EUA has approved some AG-RDTs and high specificity has been observed in antigen-based detection methods, the sensitivity of AG-RDTs is low (Table 3) . Seo et al. studied a biosensor based on a field-effect transistor, which can detect SARS-CoV-2 with ZHANG ET AL.

| 1361 a concentration of 2.42 × 10 2 copies/ml in clinical specimens in approximately 3 min without sample pretreatment. 39 Antigen detection is suitable for early detection and has the characteristics of rapid detection without expensive equipment and laboratories, and the false positive rate of detection results is also low. antibodies in blood sources such as serum, plasma, or whole blood based on the immunological principle of antigen-antibody specific binding. 41 Given that the typical time of detecting the SARS-CoV-2 immune response is approximately 1-2 weeks, serological detection has a limited diagnostic effect on SARS-CoV-2 in the acute stage of disease, but it may be valuable once the immune response occurs in time. Commonly used methods include enzyme-linked immunosorbent assay, chemiluminescence (CLIA), and LFIA. According to a meta-systematic evaluation and meta-analysis published in the past, we summarized the sensitivity and specificity of the three methods as follows ( Table 4 ). The sensitivity of the three methods is related to the duration of onset, and the sensitivity of the three methods in the third week of onset is higher than that in the first 2 weeks. The CLIA method is the most sensitive of the three serological detection methods, and the specificity is not much different.

The diagnosis of SARS-CoV-2 is mainly based on nucleic acid detection and gene sequencing. However, nucleic acid detection will be affected by many factors, such as the infection cycle of patients, specimen types, specimen collection methods, specimen preserva- 

Under the background of the current epidemic situation in COVID-19, it is still the most important task to develop diagnostic reagents and methods with high sensitivity, high specificity, low cost, easy operation, and short time. Figure 2 is the mode diagram of the detection technology summarized in this study. Real-time fluorescence quantitative PCR has become the gold standard for SARS-CoV-2 detection because of its high sensitivity and strong specificity.

However, it is easy to have false negatives due to various factors.

How to solve the problem of false negatives is worth considering.

LAMP and CRISPR/Cas have apparent advantages in nucleic acid quantification and sensitivity. Sequencing technology is reliable, but it needs expensive instruments and high-tech personnel, so it is not widely used at the grassroots level. Antibody detection assists the detection of SARS-CoV-2 to some extent. In addition, facing the problem of virus traceability, countries worldwide need to work together to develop new detection methods and technologies that are more efficient, more accurate, small, and cheap and provide genuinely effective prevention and control measures for all humankind to control the COVID-19 epidemic.

This study was supported by the key project of provincial ministry coconstruction, Health Science and Technology project plan of F I G U R E 2 Methods and techniques for detecting or identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in novel coronavirus. CLIA, chemiluminescence; ELISA, enzyme-linked immunosorbent assay; LFIA, lateral flow immunoassay; NEAR, nick enzyme-assisted reaction; RT-LAMP, reverse transcription-loop-mediated isothermal amplification; RT-PCR, reverse transcriptionpolymerase chain reaction; RT-RPA, reverse transcription-recombinase polymerase amplification; TMA, transcription-mediated amplification

Key Laboratory of Women's Reproductive Health Research of Zhejiang Province (No. ZDFY2020-RH-0006), the National Natural Science Foundation of China (Grant/ Award Numbers: U20A20351), and Key Research and Development Plan of Zhejiang Province

Covid-19 and kidney transplantation

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The authors declare that there are no conflict of interests.

Qing Ye had the idea for and designed the study and took responsibility for the integrity of the data and the accuracy of the data analysis.Hanyan Meng and Huihui Liu contributed to the writing of the report.Xiucai Zhang and Hanyan Meng contributed to the critical revision of the report. Xiucai Zhang and Hanyan Meng contributed to the statistical analysis. All authors contributed to data acquisition, data analysis, or data interpretation and reviewed and approved the final version.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0002-6756-0630