摘要
新型冠状病毒(2019-nCoV)席卷全球,目前仍处于高发阶段.高灵敏、高准确率的核酸分子诊断技术及方法,成为全球攻关热点.本研究基于微滴式数字PCR检测技术,利用巢式PCR原理,开发了检测试剂,建立了检测方法.对333个新型冠状病毒肺炎临床患者及所在环境样本进行了应用检测,与实时荧光定量PCR(RT-PCR)进行对比分析.本研究建立的微滴式数字PCR方法的线性范围在25~5 copies/μL之间,最低检出限可达0.5 copies/μL,高于市售实时荧光定量PCR检测技术的2~3个数量级.在333例样本中,环境样本197例中检测出7例阳性,人源样本136例中检测出9例阳性,检测准确度可达100%,并且检测出了RT-PCR无法检测的环境中微量病毒残留.本研究建立的数字PCR检测方法具有高度特异性,最低检出限远远低于RT-PCR.对疑似2019-nCoV感染者、早期感染者以及环境中微量病毒灵敏准确检测具有重大意义.
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 dd PCR).dd PCR 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 3332019-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.
作者
孙婷婷
姜艳芳
宫平
于源华
Tingting Sun;Yanfang Jiang;Ping Gong;Yuanhua Yu(School of Sciences,Changchun University of Science and Technology,Changchun 130022,China;The First Bethune Hospital of Jilin University,Changchun 130022,China)
出处
《科学通报》
EI
CAS
CSCD
北大核心
2021年第13期1653-1662,共10页
Chinese Science Bulletin
基金
吉林省新冠肺炎疫情防控应急科研攻关专项(20200901014SF)资助。
作者简介
联系人:于源华,E-mail:2019200032@mails.cust.edu.cn。
