Detecting and isolating false negatives of SARS-CoV-2 primers and probe sets among the Japanese Population: A laboratory testing methodology and study

The global COVID-19 pandemic has spread across various continents in diverse methods and speed while opening up discussion for technological and scientific questions pertaining methodology of testing accuracy among diverse viral strains. On the issue of testing sensitivity and accuracy, RT-PCR is commonly viewed as a standard testing protocol globally as the predominant accurate testing method as opposed to other rapid testing methods. However, each country9s infectious disease authority has established its own primers/probe sets guidelines and protocols, thereby, the global testing community lacks a "Standardized Universal Primer(s)" (SUP) that is foolproof for the COVID-19 patients among various populations today. As a result, RT-PCR testing accuracy and results may vary depending on which primer was used, most likely resulting in false negative and/or false positive calls associated with RT-PCR testing. In this study, a comparative study between primers from different countries9 disease control centers was conducted. 11,652 samples from Japanese population were tested for SARS-CoV-2 positive using recommended RT-PCR primers/probe sets from Japan National Institute of Infectious Disease (NIID) and US Centers for Disease Control and Prevention (CDC). Results: Of the 102 positive samples, 17 samples (16.7% of total positives) showed inconsistent results when tested for the following primers: JPN-N2, JPN-N1, CDC-N1, and CDC-N2. In addition, CDC recommended primer/probe sets showed relatively higher sensitivity and accuracy among the primer/probe sets used for the detection of SARS-CoV-2 positive clones. Conclusion: Due to the inconsistency in the positive/negative results for JPN-N2, JPN-N1, CDC-N1, and CDC-N2 primer/probe sets, SARS-CoV-2 samples run via RT-PCR will result in false negative/positive subjected to differences in virus mutation in a specific sequence region targeted by a primer. This outcome shows that the use of JPN-N2 primer combined with CDC-N2 primer produces the most effective result to reduce false negatives in Japan region. Furthermore, adding CDC-N1 will result in reducing false negatives, but also false positives. Further investigation remains open for confirmation whether similar irregular pattern occur with primers targeting other regions such as E or ORF1ab in order to isolate false negatives and/or positives in future PCR testing for COVID-19. Keywords: COVID-19, SARS-CoV-2, RT-PCR testing, RT-PCR performance, Genomic variants, Primers, Probe sets, Sensitivity.