基于实时荧光定量PCR技术的猪伪狂犬病活疫苗含毒量评价

doi:10.16431/j.cnki.1671-7236.2019.04.021

摘要/Abstract

摘要：

为定量分析贵州省临床用猪伪狂犬病（pseudorabies，PR）活疫苗中病毒含量，根据GenBank中公布的伪狂犬病病毒（pseudorabies virus，PRV）gB基因序列（登录号：M17321.1）设计1对特异性引物，建立实时荧光定量PCR方法，并对来自6个不同厂家的猪伪狂犬病活疫苗进行含毒量分析。结果显示，试验成功建立了可用于PRV检测的实时荧光定量PCR方法，标准曲线中标准品各稀释度质粒拷贝数与Ct值呈良好的线性关系，标准曲线方程为：Y=-0.97X+33.66（R²=0.999），该方法最低检测病毒含量为3.19×10¹拷贝/μL，敏感性高且具有良好的重复性和特异性。应用所建立的实时荧光定量PCR方法对贵州省临床用6个厂家生产的猪伪狂犬病活疫苗的病毒含量进行检测，结果显示，6种市售猪伪狂犬病活疫苗（A~F）中病毒含量分别为1.67×10⁷、4.83×10⁵、2.64×10⁶、4.27×10⁷、3.39×10⁶和3.68×10⁵拷贝/μL，来自不同厂家的疫苗病毒含毒量存在明显差异，最大差异可达116倍，其中来自A、D厂家的两种猪伪狂犬病活疫苗具有较高的含毒量。本试验所建立的实时荧光定量PCR方法可用于猪伪狂犬病活疫苗病毒含量的快速检测和评价，对猪伪狂犬病活疫苗的质控和临床用疫苗选择具有一定的指导意义。

关键词: 猪伪狂犬病; 疫苗; 病毒含量; 评价

Abstract:

In order to quantitatively analyze the virus content of clinical live pseudorabies (PR) vaccine in Guizhou province,a pair of specific primers was designed according to the sequence of pseudorabies virus (PRV) gB gene in GenBank (accession No.:M17321.1),a Real-time quantitative PCR method was established,and the virus content of live PR vaccine from 6 different manufacturers was analyzed.The results showed that a Real-time quantitative PCR method was successfully established for detection of PRV.There was a good linear relationship between the number of plasmid copies diluted in the standard curve and Ct value,the standard curve equation was Y=-0.97X+33.66 (R²=0.999).The method had a minimum detection virus content of 3.19×10¹ copies/μL,high sensitivity,good repeatability and specificity.The Real-time quantitative PCR method was applied to detect the viral content of live PR vaccine produced by 6 manufacturers in Guizhou province.The results showed that the virus content of 6 live PR vaccine (A-F) sold in 6 markets were 1.67×10⁷,4.83×10⁵,2.64×10⁶,4.27×10⁷,3.39×10⁶ and 3.68×10⁵ copies/μL,the viral content of the vaccine from different manufacturers was significantly different,the biggest difference could be 116 times.Two kinds of live PR vaccine from A and D manufacturers had higher viral content.The Real-time quantitative PCR method established in this experiment could be used for rapid detection and evaluation of the viral content of live vaccine against porcine PR,it had a certain guiding significance for the quality control of the live vaccine of PR and the selection of clinical vaccine.

Key words: pseudorabies (PR); vaccine; virus content; evaluation

中图分类号:

S852.65⁺9.1

王柏林, 周怡, 何玲, 杨美, 程振涛. 基于实时荧光定量PCR技术的猪伪狂犬病活疫苗含毒量评价[J]. 《中国畜牧兽医》, 2019, 46(4): 1135-1142.

WANG Bolin, ZHOU Yi, HE Ling, YANG Mei, CHENG Zhentao. Evaluation of Virus Content of Live Pseudorabies Vaccine Based on Real-time Quantitative PCR[J]. , 2019, 46(4): 1135-1142.

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