Rapid displacement of SARS-CoV-2 variants within Japan correlates with cycle threshold values on routine RT-PCR testing

BackgroundThe rapid spread of SARS-CoV-2 worldwide has led to the emergence of new variants due to the presence of mutations that alter viral characteristics, but there have been few studies on trends in viral lineages in Japan, an island country. We hypothesized that changes in cycle threshold (Ct) values on reverse transcription polymerase chain reaction (RT-PCR) reflect the prevalent variants during a given period. MethodsWe performed next-generation sequencing of positive samples to identify the viral lineages in Japan in 2021 and compared variant prevalence with the average Ct values on routine RT-PCR using 4 primer sets. ResultsBased on 3 sequencing runs, the highly transmissible Alpha variant, which prevailed over other lineages, such as R.1, from April 2021, was dominated by the even stronger Delta variant between July and August 2021 in Japan. The decrease in our routine RT-PCR Ct values with 4 primer sets correlated with these fluctuations in lineage prevalence over time. ConclusionsWe confirmed that our RT-PCR protocol reflects the trends in SARS-CoV-2 variant prevalence over time regardless of sequence mutation. This may aid in the tracking of new variants in the population.

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

ObjectivesIn this study, a comparative study between primers from Japans and USs disease control centers was conducted. As further investigation, virus sequence alignment with primers oligonucleotide was analyzed. Design or methods11,652 samples from Japanese population were tested for SARS-CoV-2 positive using recommended RT-PCR primer-probe sets from Japan National Institute of Infectious Disease (NIID) and US Centers for Disease Control and Prevention (CDC). ResultsOf the 102 positive samples, 17 samples (16.7% of total positives) showed inconsistent results when tested simultaneously for the following primers: JPN-N2, JPN-N1, CDC-N1, and CDC-N2. As a result, CDC recommended primer-probe sets showed relatively higher sensitivity and accuracy. Further virus sequence alignment analysis showed evidences for virus mutation happening at primers binding sites. ConclusionsThe inconsistency in the RT-PCR results for JPN-N1, JPN-N2, CDC-N1, and CDC-N2 primer-probe sets could be attributed to differences in virus mutation at primers binding site as observed in sequence analysis. The use of JPN-N2 combined with CDC-N2 primer produces the most effective result to reduce false negatives in Japan region. In addition, adding CDC-N1 will also help to detect false negatives.