A Novel Primer Probe Set for Detection of SARS-CoV-2 by Sensitive Droplet Digital PCR

Wang, F.; Pervaiz, U.; Tian, H.; Gahdary, M. O. A. O. A.; Hamid, M. A. M.; Wang, D.

Background: The current increase in the spread of (SARS-CoV-2) critically needs a multitarget diagnostic assays to promote analytical sensitivity to facilitate the public health actions. Objective: The aim of this study was to develop a new primer-probe set targeting N gene of SARS-CoV-2 to improve the sensitivity for detection of COVID-19 Corona Virus Disease 2019 in multiplex rRT-PCR (Reversetranscript Realtime PCR) and ddPCR (Droplet Digital PCR). Results: We designed primers/probes set N(LZU3) targeting the N gene of 2019-nCov and proved its sensitivity in both rRT-PCR and ddPCR. When the quantity of template was 105 copies/reaction, the mean Ct value of N(LZU3) was 32.563, the detection rate was 91.7%. If the quantity of template was 52.5 copies/reaction, the mean Ct value of N(LZU3) was 33.835, and the detection rate was 83.3%, which were similar with that of N(CDC) and N(USA). The calculated lower limit of detection (LOD) of the new primer-probe set N(LZU3) used in rRT-PCR was 118 copies/reaction. We also did one-step ddPCR for detection the same serial dilution of RNA template. It shows good linearity for primer/probe sets N(LZU3). The calculated lower limit of detection (LOD) of N(LZU3) was 22.4 copies/reaction, which was 1.12 copies/ul. Conclusion: The novel primer-probe set(LZU3) targeting N gene of SARS-CoV-2 could be both used in rRT-PCR and ddPCR with better sensitivity, furthermore, ddPCR method had higer sensitivity than rRT-PCR, hence it could significantly improve SARS-CoV-2 detection efficiency in low virus load and asymptomatic infection.