Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background:</jats:bold> In absence of effective vaccines, infection prevention and control of SARS-CoV-2 through diagnostic testing and quarantine is critical. Early detection and differential diagnosis of respiratory infections increases the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis with high sensitivity. However, the highest specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, a large amount of recent evidence indicates that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” may be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false negative results. Moreover, the current used mixed sample nucleic acid detection is helpful to seek out the early community transmission of SARS-CoV-2, but the detection kit needs ultra-high detection sensitivity. <jats:bold>Methods: </jats:bold>From January to March 2020, 10 confirmed specimens of 2019-nCoV were recruited from three 2019-nCOV nucleic acid testing center. Five different amplification kits with three different primers and probes sources were selected. RT-PCR and continuous amplification of nasopharyngeal and oropharyngeal swabs and environmental specimens were performed to validate the sensitivity of the commercially available Nucleic acid test kits. <jats:bold>Results:</jats:bold> The results showed that ORF1ab gene can still be reported as positive at 1:10 dilution and the N gene even at 1:40 dilution with kit-1 through the verification of multiple positive samples, While other kits have less sensitive. The results in the suspicious range of weakly positive nasopharyngeal and oropharyngeal swabs and environmental specimens could be reported as positive after another re-amplification.<jats:bold>Conclusions:</jats:bold> Through evaluate the sensitivity of different nucleic acid detection kits, this study provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.</jats:p>

Genetic Diversity Among SARS-CoV2 Strains in South America may Impact Performance of Molecular Detection

“…As mentioned earlier, this is the reason why the occurrence of false-negative results in the context of ongoing epidemics should be taken cautiously. Evidence from Europe on the occurrence of false-negative results for N-gene-based assays support our in silico findings [39,40]. Despite no present evidence on false-negative results for RdRp gene-based assays, its inherent mutation rate (due to environmental pressure and its role as a virulence factor), and considering how this gene showed low sensitivity [41] is an aspect that deserves further investigation.…”

MicroRNA of N‐region from SARS‐CoV‐2: Potential sensing components for biosensor development

“…These sequences are the RdRP gene, E gene, N gene, ORF1ab gene, and S gene. 53,54 N-gene is the most abundantly expressed transcript in SARS-CoV-2-infected cells at the early stage of infection, thus, enhances the efficiency of viral RNA transcription that is essential for viral replication. 55,56,57 This suggests that the N-gene is one of the best targets for high sensitivity detection of SARS-CoV-2 infection.…”

“…It also exhibits the most sensitive assay target even in the low concentration of the virus in detecting SARS-CoV-2 using RT-PCR and RT-Lamp assay. 45,53 The main factor that N-gene is more preferred as the target for detecting SARS-CoV-2 virus is that the N gene is more conserved, has fewer nucleotide variation, and lower mutation rate compared with the ORF1ab, thus making the detection of the N-gene more stable, which could partially explain why the N-gene was most often detected in the test results. 58 Except for the N gene, due to these variations' potential to interact with both diagnostic tests and antiviral therapies such as Remdesivirir, a gene mutation in the RdRp gene has been well established.…”

Evaluation of deep learning for COVID‐19 diagnosis: Impact of image dataset organization

“…However, it has low sensitivity of 60%–70% and takes a relatively long time to obtain the test result 4–6 . RT‐PCR has been improved following the efforts of many researchers; 7–9 nevertheless, it continues to have limitations. Furthermore, there are shortages of RT‐PCR test kits and reagents to deal with the many tests required and the test is expensive.…”