马疱疹病毒1型QuantStudioTM 3D数字PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2020.05.002

摘要/Abstract

摘要： 试验旨在建立一种高灵敏性的马疱疹病毒1（EHV-1）3D 数字PCR（3D-dPCR）检测方法，对EHV-1病毒含量较低的样品能准确定量检出，实现马鼻肺炎早期诊断及预防。根据EHV-1糖蛋白B基因保守区域设计特异性引物和探针，优化3D-dPCR反应体系中引物探针浓度和退火温度，对该方法进行灵敏性、特异性、重复性分析，建立了EHV-1的3D-dPCR方法。本研究建立的3D-dPCR方法，引物和探针最佳浓度分别为 0.4和0.4 μmol/L，最佳退火温度为60 ℃，该方法绝对定量曲线的R²=0.998，线性关系良好，与实时荧光定量PCR方法相比灵敏度高10倍左右，最低检出限为5.83拷贝/μL；批内和批间重复性试验变异系数均<3.2%；与EHV-4、马泰勒虫、马病毒性动脉炎的核酸无交叉反应；通过对123份临床样品进行3D-dPCR检测，结果显示，3D-dPCR方法阳性检出率为66.7%，高于世界动物卫生组织（OIE）中EHV-1的实时荧光定量PCR方法阳性检出率64.2%。3D-dPCR方法对病毒含量较高的样品与实时荧光定量PCR结果一致，对病毒含量较低的样品敏感性更高，能有效检出可疑样品。本试验结果表明，建立的3D-dPCR方法检测低拷贝数的临床样品时灵敏度高、特异性强、重复性好，可用于EHV-1的准确定量检测。

关键词: 马疱疹病毒1型(EHV-1); 3D 数字 PCR(3D-dPCR); 绝对定量

Abstract: The purpose of this study was to establish a highly sensitive 3D digital PCR (3D-dPCR) method for the detection of equine herpesvirus 1 (EHV-1),which could accurately and quantitatively detect the samples with low EHV-1 content and realize the early diagnosis and prevention of equine rhinopneumonia.According to the conserved region of EHV-1 glycoprotein B gene,we designed specific primers and probes,optimized the concentration and annealing temperature of primers in the 3D-dPCR reaction system,analyzed the sensitivity,specificity and repeatability of this method,and established the 3D-dPCR method of EHV-1.In this study,the best concentration of primer and probe of 3D-dPCR was 0.4 and 0.4 μmol/L respectively,the best annealing temperature was 60 ℃,R² of the absolute quantitative curve of the method was 0.998,the linear relationship was good,the sensitivity was about 10 times higher than that of Real-time PCR,and the minimum detection limit was 5.83 copies/μL.There was no cross reaction with EHV-4,Theileria equi and the nucleic acid of equine arteritis.The results showed that the positive rate of 3D-dPCR was 66.7%,which was higher than that of Real-time PCR for EHV-1 in OIE (64.2%).The results of 3D-dPCR were consistent with those of Real-time PCR,and the sensitivity of 3D-dPCR to the samples with low virus content was higher,which could effectively detect suspicious samples.The results showed that the established 3D-dPCR method was more sensitive,specific and reproducible for the detection of clinical samples with low copy number,and could be used for the accurate and quantitative detection of EHV-1.

Key words: equine herpesvirus type 1 (EHV-1); 3D digital PCR (3D-dPCR); absolute quantification

中图分类号:

S852.65

雷程红, 陈凯云, 徐新峰, 胡都斯·艾尔肯, 徐军, 白梅花, 史茜, 肖媛媛, 王科珂. 马疱疹病毒1型QuantStudio^TM 3D数字PCR检测方法的建立[J]. 中国畜牧兽医, 2020, 47(5): 1299-1306.

LEI Chenghong, CHEN Kaiyun, XU Xinfeng, HUDUSI·Aierken, XU Jun, BAI Meihua, SHI Qian, XIAO Yuanyuan, WANG Keke. Establishment of QuantStudio^TM 3D Digital PCR Method for Detection of Equine Herpesvirus Type 1[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1299-1306.

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