数字PCR技术检测感染者尿液中SARS-CoV-2的方法研究

Study on the detection of SARS-CoV-2 in the urine of infected persons by droplet digital PCR

目的建立灵敏和准确可靠的数字PCR反应体系,开发简单高效的临床检测SARS-CoV-2的分析方法,通过提高检出限,为大规模爆发的传染性疾病快速诊断提供解决方案。方法提取12例病人咽拭子和尿液样本中的总RNA,以反转录后的cDNA为模板配制反应体系,经过油滴发生器将反应体系分割成油滴并进行PCR扩增,再结合荧光读取,分析检测样本中阳性核酸片段的数量。制备包含SARSCoV-2病毒核酸片段共2983 bp的阳性质粒,以10倍系列稀释液为模板进行RT-qPCR反应,根据标准质粒扩增结果制备标准曲线,通过标准曲线计算未知样本目的片段数量,比较ddPCR与RT-qPCR检测结果的准确性。以尿液为实验材料,检测阳性感染者尿液中病毒核酸拷贝数。结果12份阳性尿液标本检出率为75%,最低检出限为5.4 copies/μL,与常规RT-qPCR相比灵敏度显著提高。结论ddPCR方法准确、快速,且灵敏度高,可以满足尿液标本中低拷贝核酸的快速检出。

Objective The objective of the present study is to establish a sensitive,accurate and reliable polymerase chain reac⁃tion(PCR)platform,to develop a simple and effective analytical method to measure the SARS-CoV-2 clinically,and to provide solution of rapid diagnosis in case of outbreaks of infectious diseases through increasing the sensibility of detection limit.Methods The total RNA was extracted from throat swabs and urine of 12 infected people,respectively.The cDNA was used as templates and the PCR pro⁃gram was carried out after the generation of the droplets.The concentration of positive fragments were calculated by the droplet analyzer.Integrated with a 2983 bp fragment of SARS-CoV-2,the pUC57 plasmid was diluted by multiples of 10 and used as templates for RT qPCR.The standard curve was made according to the results of the amplification of the standard plasmid.The original concentration in specimen was calculated according to the standard curve.The veracity of the measurements of both ddPCR and RT-qPCR were com⁃pared,respectively.The quantity of the original copies in urine was measured through ddPCR method.Results The detection rate of 12 positive urine samples was 75%,and the detection limit was 5.4 copies/μL,the detection sensitivity of that was obviously higher in com⁃parison with RT-qPCR method.Conclusions ddPCR is accurate,rapid and sensitive,which can meet the requirements of rapid detec⁃tion of low copy nucleic acid in urine samples.