The development of a droplet digital PCR for accurate detection of SARS-CoV- 2 by simultaneous determination of dual gene targets

Abstract Real-time fluorescent quantitative PCR is considered as the gold standard for diagnosing COVID-19 worldwide now. But RT-PCR which could yield semi-quantitative results only, and the sensitivity of this method is usually not sufficient to discriminate patients in early stages of infection or with a very low viral load. So, the false-negative phenomenon often brings difficulties to epidemic prevention and diagnosis. More accurate and robust determine method are required for accurate SARS-CoV-2 diagnosis. To offer an attractive platform for SARS-CoV-2 RNA quantification, we report a droplet digital PCR technique for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. Two pairs of primer and two double-quenched probes targeting the region of ORF1ab and N protein coding genes of SARS-CoV-2 were designed to develop ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested by standard cDNAs and clinical specimens. First, we found the optimal annealing temperature was 53.6℃, the optimal primer concentration was 500 nM per reaction. When both probes of ORF1ab and N protein are applied simultaneously, their concentrations are100 nM and 50 nM, respectively. The optimal concentration of SARS-CoV-2 standard cDNA ranged from 1×103 to 1×104 copies/µL. And under this condition, the trend line for the measured concentration is Y = 0.9978x-0.4435. Further, the ddPCR were approximately 10 folds greater sensitivity than RT-qPCR assay. The established ddPCR assay with both probes showed to be a highly effective, accurate and reliable method for the sensitive detection of SARS-CoV-2. And the ddPCR method is the most potential and valuable method for clinical detection of SARS-CoV-2 virus.