鸡传染性喉气管炎TaqMan探针实时荧光定量PCR检测方法的建立与应用
Establishment and application of TaqMan probe real-time fluorescence quantitative PCR method for detection of infectious laryngotracheitis in chickens
张相霞 1王晓梦 2张帅2
作者信息
- 1. 山东省平邑县畜牧发展促进中心,山东临沂 273300
- 2. 山东农业大学动物科技学院,山东泰安 271018
- 折叠
摘要
为了建立一种鸡传染性喉气管炎病毒(ILTV)的快速诊断方法,基于ILTV gB基因设计特异性探针和引物,建立一种基于TaqMan探针的实时荧光定量PCR(qPCR)方法.结果显示,ILTV qPCR方法的标准曲线为y=-3.892 9x+44.607,线性相关值(R2)为0.998 8.该方法的最低检测限为1.0× 10 copies/μL,批内与批间重复性检测的变异系数均小于5.64％,且与其他鸡源病原体无交叉反应.临床样本检测结果显示,该qPCR方法的检测阳性率为98.7％,而普通PCR的检测阳性率为92.61％.以上结果表明,该方法灵敏度高、特异性强、重复性好,可用于ILTV的疫病监测和流行病学调查,为ILTV感染的预防和诊断提供了有力工具.
Abstract
To establish a rapid diagnostic method for chicken infectious laryngotracheitis virus(ILTV),specific probes and primers were designed based on the ILTV gB gene,and a real-time fluorescent quantitative PCR(qPCR)method based on the TaqMan probe was established.The results showed that the standard curve of the ILTV qPCR method was y=-3.892 9x+44.607 with a linear correlation value(R2)of 0.998 8.The lowest detection limit of the method was 1.0× 10 copies/μL,and the coefficients of variation for both intra-and inter-batch reproducibility were less than 5.64％,and there was no cross-reactivity with other chicken-derived pathogens.The test results of the clinical samples showed a positive detection rate of 98.7％for the qPCR method compared to 92.61％for conventional PCR.The above results indicate that the method is sensitive,specific,and reproducible,and can be used for epidemic surveillance and epidemiologi-cal investigation of ILTV,providing a powerful tool for the prevention and diagnosis of ILTV infection.
关键词
鸡传染性喉气管炎病毒/gB基因/实时荧光定量PCR/TaqMan探针Key words
chicken infectious laryngotracheitis virus/gB gene/real-time fluorescence quanti-tative PCR/TaqMan probe引用本文复制引用
基金项目
江苏省科技计划(BE2021332)
江苏省科技计划(BE2022318)
江苏省农业科技自主创新项目(CX231004-3)
出版年
2024
国家科技期刊平台
NSTL
万方数据