伪狂犬病病毒微滴数字PCR定量检测方法的建立

doi:10.11843/j.issn.0366-6964.2017.09.015

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (9): 1705-1710.doi: 10.11843/j.issn.0366-6964.2017.09.015

伪狂犬病病毒微滴数字PCR定量检测方法的建立

陈亚娜¹, 王静¹, 訾占超¹, 亢文华¹, 汪葆玥¹, 倪建强¹, 原霖^1,2*

1. 中国动物疫病预防控制中心, OIE猪繁殖与呼吸综合征参考实验室, 北京 102618;
2. 中国农业大学动物医学院, 北京 100193

收稿日期:2017-03-06 出版日期:2017-09-23 发布日期:2017-09-14
通讯作者: 原霖,E-mail:6428949@163.com
作者简介:陈亚娜(1993-),女,河北沧州人,学士,主要从事动物疫病诊断技术研究,E-mail:1245471514@qq.com
基金资助:
科技部科技基础性工作专项（2013FY113300-6）

Development of Droplet Digital PCR for the Detection of Pseudorabies Virus

CHEN Ya-na¹, WANG Jing¹, ZI Zhan-chao¹, KANG Wen-hua¹, WANG Bao-yue¹, NI Jian-qiang¹, YUAN Lin^1,2*

1. OIE Reference Laboratory for Porcine Reproductive and Respiratory Syndrome, China Animal Disease Prevention and Control Center, Beijing 102618, China;
2. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Received:2017-03-06 Online:2017-09-23 Published:2017-09-14

摘要/Abstract

摘要：

为了建立伪狂犬病病毒（PRV）微滴数字PCR（droplet digital PCR，ddPCR）的检测方法，以伪狂犬病病毒gB基因为靶基因，建立了可对其准确定量的微滴数字PCR方法。对ddPCR反应中的引物浓度、探针浓度、退火温度进行了优化。对建立的ddPCR方法的特异性和敏感性也进行测定，并且应用于临床样品的检测。结果表明，ddPCR的最佳引物浓度为0.9 μmol·L^-1，探针浓度为0.25 μmol·L^-1，退火温度为60℃。ddPCR方法线性相关系数（R²）为0.998，显示呈良好的线性关系。检测限为6.1 copies·μL^-1，比荧光定量PCR（Real Time PCR，qPCR）的检测限低，变异系数为2.81%，与其他病毒无交叉反应。通过对临床样品的检测，与病毒分离比较，ddPCR方法的敏感性为87.5%，特异性为96.2%，符合率为97%。结果表明新建立的ddPCR方法，敏感性高、特异性强，可用于伪狂犬病病毒的定量检测。

关键词: 伪狂犬病病毒, 微滴数字PCR, 检测方法

Abstract:

A droplet digital polymerase chain (ddPCR) detection method, based on gB gene of pseudorabies virus (PRV) was developed to improve the detection and quantification of PRV. The concentration of primer, probe concentration and annealing temperature in ddPCR reaction were optimized. The specificity and sensitivity of the established ddPCR method were also determined and applied to the detection of field samples. In results,the optimal primer concentration was 0.9 μmol·L^-1, the probe concentration was 0.25 μmol·L^-1, and the annealing temperature was 60℃ for ddPCR.The R² value of ddPCR standard curve was 0.998,showed ddPCR had a good linear response. The Limit of Detection of ddPCR was 6.1 copies·μL^-1, lower than that of fluorescence quantitative PCR (qPCR). The coefficient of variation was 2.81%,the ddPCR was specific for detecting PRV. Field samples were detected by ddPCR, qPCR and virus isolation. Compared with the virus isolation, the sensitivity of ddPCR method was 87.5%, specificity was 96.2%, and the coincidence rate was 97%. The results indicate that the new ddPCR assay provides a specific, sensitive tool for quantitative detection of PRV.

Key words: pseudorabies virus, droplet digital PCR, detection

中图分类号:

S852.659.1

陈亚娜, 王静, 訾占超, 亢文华, 汪葆玥, 倪建强, 原霖. 伪狂犬病病毒微滴数字PCR定量检测方法的建立[J]. 畜牧兽医学报, 2017, 48(9): 1705-1710.

CHEN Ya-na, WANG Jing, ZI Zhan-chao, KANG Wen-hua, WANG Bao-yue, NI Jian-qiang, YUAN Lin. Development of Droplet Digital PCR for the Detection of Pseudorabies Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1705-1710.

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伪狂犬病病毒微滴数字PCR定量检测方法的建立

Development of Droplet Digital PCR for the Detection of Pseudorabies Virus

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