Detection of microamounts of novel coronavirus residues in environment by digital PCR

Abstract

<p indent="0mm">2019 novel coronaviruses (2019-nCoV) swept the world and are still at a high incidence stage. Highly sensitive and accurate nucleic acid molecular diagnostic techniques and methods have become a hot spot of global concern. 2019-nCoV is a kind of RNA virus. At present, there are many methods to detect virus RNA, including real-time fluorescence quantitative PCR (RT-PCR), colloidal gold detection, chemiluminescence detection, and so on. Colloidal gold detection and chemiluminescence detection need to use monoclonal antibodies against 2019-nCoV, but there are some problems in the preparation of 2019-nCoV monoclonal antibodies, such as long cycle, complex preparation and easy pollution. So the above two methods are not easy to be established for the detection of 2019-nCoV. The superiority of PCR technology is widely used in auxiliary medical clinical diagnosis, customs inspection and quarantine, the development of new agriculture, national defense military defense, and basic research of bioscience, and so on. However, the quantification of the target DNA fragment in RT-PCR is still relatively quantitative. Because this technique mainly depends on CT value and standard curve to quantify the target DNA in the experiment, which affects the amplification efficiency of the whole system, and the sensitivity and accuracy of the reaction system are greatly affected. Because the 2019-nCoV virus is highly contagious, the detection methodology is required to be highly sensitive, which is in line with the characteristics of digital PCR (droplet digital PCR system, referred to as ddPCR). ddPCR has the advantages of absolute quantification, high specificity, high sensitivity and strong interference ability, which can better combine molecular biology and medicine closely, and has a great advantage in the detection of 2019-nCoV. In this study, based on droplet digital PCR detection technology, the detection reagent and method were established using the principle of nested PCR. The Saliva of 333 2019-nCoV patients and their environmental samples were detected and compared with real-time PCR (RT-PCR). The linear range of the droplet digital PCR method established in this study was between 25–5 copies/µL, and minimum detection limit was 0.5 copies/µL, which was 2–3 orders of magnitude higher than that of the commercial real-time fluorescent quantitative PCR detection technology. In 333 samples, 7 were positive in 197 environmental samples, and 9 were positive in 136 human samples. The detection accuracy was 100%, and the environmental micro-virus residues that could not be detected by RT-PCR were detected. The digital PCR detection method established in this study was highly specific, and the minimum detection limit was far lower than that of RT-PCR. It is of great significance for sensitive and accurate detection of 2019-nCoV infection, early infection and environmental microvirus.