Abstract:

The goal of this research is to develop a fast and real-time fluorescent reverse-transcription recombinase polymerase amplification method（RT-RPA）method for detecting and monitoring SARS-CoV-2. Two pairs of RPA primers/probe were initially designed based on the conserved sequences of specific S gene and Ngene of SARS-CoV-2. Meanwhile,SARS-CoV-2 virus-like particles based on the recombinant plasmids containing targeted N gene fragment was developed to offer positive material for molecular biological detection. Finally a set of RPA primers and probes targeting S gene and N gene was selected for developing fluorescent RT-RPA test strips. The analytical sensitivity of this method was about 10 copies/reaction of S gene and 10² copies/reaction of N gene. The RT-RPA could not amplify templates of H1N1 influenza virus,PEDV,TGEV,FMDV,PPRV and RV,demonstrating its high specificity. The established method was revealed to exclusively detect SARS-CoV-2 in foods,animal tissues and clinical samples,suggesting that the RT-RPA method is fast（20 minutes）,double-targeting,sensitive,easy-operating and not depending on the expensive equipment,which makes it an effective approach for the rapid detection of SARS-CoV-2 in foods,animal products and clinical samples.

Key words: SARS-CoV-2, fluorescent RT-RPA, virus-like particles