畜牧兽医学报2017，Vol.48Issue(9)：1705-1710,6.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
Development of Droplet Digital PCR for the Detection of Pseudorabies Virus
摘要
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.关键词
伪狂犬病病毒/微滴数字PCR/检测方法Key words
pseudorabies virus/droplet digital PCR/detection分类
农业科技引用本文复制引用
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):1705-1710,6.基金项目
科技部科技基础性工作专项(2013FY113300-6) （2013FY113300-6）
