牛病毒性腹泻病毒一步法微滴数字PCR检测方法的建立与初步应用

Establishment and preliminary application of a one-step reverse transcriptase droplet digital PCR detection method for bovine viral diarrhea virus

为建立牛病毒性腹泻病毒(BVDV)一步法微滴数字PCR(RT-ddPCR)方法,本研究在一步法实时荧光定量PCR(RT-qPCR)检测方法的基础上,设计BVDV的特异性引物和探针,对RT-ddPCR试验中的反转录酶、退火温度、引物和探针浓度以及检测方法的反应条件进行优化。同时对其特异性、敏感性、重复性进行评估。结果显示,建立的RT-ddPCR方法最佳反转录体系为商品化一步法数字PCR试剂盒配套试剂、引物和探针终浓度分别为900 nmol/L和250 nmol/L,最佳退火温度为57℃;用本方法检测常见疫病病毒时结果呈阴性;本方法最低检测限为3.2 copies/μL,重复性好,且变异系数小于5%。采用RT-ddPCR和RT-qPCR对24份牛源拭子样品进行检测,检测结果显示所建方法优于RT-qPCR方法。本研究建立的RT-ddPCR方法特异性强、敏感性高、重复性好,适用于临床样品的核酸检测,为牛病毒性腹泻病毒感染的早期检测和定量诊断提供了技术支持。

In order to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)method for bovine viral diarrhea virus(BVDV),based on the one-step real-time quantitative PCR(RT-qPCR)detection method,BVDV-specific primers and Probes were designed in this study.The reverse transcriptase,annealing temperature,primer,probe concentrations and reaction conditions of the detection method in the RT-ddPCR assay are optimized.At the same time,the specificity,sensitivity and repeatability were evaluated.The results showed that the optimal reverse transcription system for the established RT-ddPCR method was a commercial one-step reverse transcriptase droplet digital PCR kit with the matching reagents,the final concentrations of primers were 900 nmol/L,the probes were 250 nmol/L and the optimal annealing temperature was 57℃.The results for other common epidemic viruses detected by this method were negative,the lowest detection limit was 3.2 copies/μL and the Repeatability was good and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR were used to test 24 bovine swab samples and the test results showed that the established method was superior to RT-qPCR.The RT-ddPCR method established in this study has strong specificity,high sensitivity and good repeatability,which is suitable for nucleic acid detection of clinical samples.The study can provide technical support for the early detection and quantitative diagnosis of bovine viral diarrhea virus infection.