SARS-CoV-2 Continuous Genetic Divergence and Changes in Multiplex RT-PCR Detection Pattern on Positive Retesting Median 150 Days after Initial Infection

Being in the epicenter of the COVID-19 pandemic, our lab tested 193,054 specimens for SARS-CoV-2 RNA by diagnostic multiplex reverse transcription polymerase chain reaction (mRT-PCR) starting in March 2020, of which 17,196 specimens resulted positive. To investigate the dynamics of virus molecular evolution and epidemiology, whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates. 7586 isolates with a high quality were further analyzed for the mutation frequency and spectrum. Lastly, we evaluated the utility of the mRT-PCR detection pattern among 26 reinfected patients with repeat positive testing three months after testing negative from the initial infection. Our results show a continuation of the genetic divergence in viral genomes. Furthermore, our results indicate that independent mutations in the primer and probe regions of the nucleocapsid gene amplicon and envelope gene amplicon accumulate over time. Some of these mutations correlate with the changes of detection pattern of viral targets of mRT-PCR. Our data highlight the significance of a continuous genetic divergence on a gene amplification-based assay, the value of the mRT-PCR detection pattern for complementing the clinical diagnosis of reinfection, and the potential for WGA and NGS to identify mutation hotspots throughout the entire viral genome to optimize the design of the PCR-based gene amplification assay.

Automated summary

This study, conducted in the epicenter of the COVID-19 pandemic, tested 193,054 specimens for SARS-CoV-2 RNA using mRT-PCR and found 17,196 positive results. Whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates to investigate virus molecular evolution and epidemiology. The results showed a continuation of genetic divergence in viral genomes, with independent mutations accumulating over time in primer and probe regions. Some of these mutations were correlated with changes in the detection pattern of viral targets for mRT-PCR. The study highlights the importance of continuous genetic divergence in gene amplification-based assays, the value of the mRT-PCR detection pattern in complementing the clinical diagnosis of reinfection, and the potential of WGA and NGS in identifying mutation hotspots to optimize PCR-based gene amplification assays.