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

Abstract

The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might 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 mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to 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.

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 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 having account this gene showed low sensitivity [41] is an aspect that deserves further investigation…”

RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus

“…Second, different probes and primer targets result in different amplification efficiencies and Ct values. 36,37 It is known that PCR amplification efficiencies critically influence the Ct values; for example, the number of viral genome copies estimated by a Ct value of 37 in PCR with an amplification efficiency of 100% is equal to that estimated by a Ct value of about 40 in PCR with an amplification efficiency of 90%. Each local laboratory in charge of diagnostic RT-qPCR testing should be aware of these differences when optimizing standard operating procedures and of the importance of regular laboratory quality checks and controls.…”

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP and human RP genes

“…In China, Orf1ab and N genes are regularly used, while N1, N2 and N3 genes were utilized in US CDC and E, N, and RdRP genes in Europe [32]. The importance of N1 and N2 primer-probes is for providing a less conservative but more sensitive than the RdRP-SARSr primer-probes especially in samples that have low viral titers [33,34]. In a recent study, where swabs from con rmed cases were taken from nasopharynx and pharynx targeting ORF1ab and N genes yielded the best sensitivity when compared to positive con rmed samples [34].…”

“…The importance of N1 and N2 primer-probes is for providing a less conservative but more sensitive than the RdRP-SARSr primer-probes especially in samples that have low viral titers [33,34]. In a recent study, where swabs from con rmed cases were taken from nasopharynx and pharynx targeting ORF1ab and N genes yielded the best sensitivity when compared to positive con rmed samples [34]. Chu et al [35] have reported two assays that had the capability to achieve a large dynamic range and recommended targeting N gene for screening and the ORF1b gene to con rm the result.…”