血清1型FAdV TaqMan探针荧光定量PCR方法的建立及应用
Development and application of a TaqMan real-time fluorescence quantitative PCR for serotype 1 FAdV
刘琳 1宋亚鹏 1刘明洋 1高文明 1陶明月 1宋晓楠 1张宜娜 1李新生1
作者信息
- 1. 河南农业大学动物医学院,河南郑州 450046
- 折叠
摘要
为特异性地检测家禽中血清1型禽腺病毒(fowl adenovirus,FAdV)的感染,本研究以血清1型FAdV保守的52K基因为模板设计引物和探针,通过矩阵法优化后建立了针对该病毒的TaqMan探针实时荧光定量PCR方法,并用该方法检测1日龄SPF鸡攻毒后咽、肛拭子排毒量及组织病毒载量.结果显示,该方法具有良好的特异性、敏感性和重复性,只能检测出血清1型FAdV,对其他血清型FAdV和其他禽类病毒均无阳性扩增;最低检测限度低至10拷贝/μL;组间和组内离散度均小于2％;标准曲线为y=-3.836x+41.791,相关系数R2=0.996,具有良好的线性关系;对SPF鸡攻毒结果显示,在攻毒后第1～14日的咽、肛拭子均可检测到血清1型FAdV,在攻毒后第8日的肝脏、肾脏、肌胃、回肠中也可检测到病毒,而腺胃和盲肠中未检出病毒.本研究为血清1型FAdV的定量检测提供了方法.
Abstract
To detect serotype 1 FAdV infection in chickens,primers and TaqMan probes were de-signed according to the conserved 52K gene of serotype 1 FAdV in this study.Real-time fluores-cence quantitative PCR optimized by the matrix method was used to detect the virus shedding of o-ral and cloacal swabs and the viral load of tissues after challenge.The results showed that the method had good specificity,sensitivity,and reproducibility;it could only detect serotype 1 FAdV,but not other serotypes of FAdV or other avian viruses.The minimum detection limit was as low as 10 copies/μL,and the inter-and intra-group dispersions were less than 2％.The standard curve was y=-3.836x+41.791,and the correlation coefficient was R2=0.996,indicating a good linear relationship.The results of the SPF chicken challenge showed that FAdV serotype 1 could be de-tected in the oral and cloacal swabs until 14 days post-infection,and the virus could also be detec-ted in the liver,kidney,gizzard,and ileum at 8 days post-infection,but not in the glandular stomach and cecum.This study provides a method for the quantitative detection of serotype 1 FAdV.
关键词
血清1型FAdV/TaqMan探针/荧光定量PCRKey words
serotype 1 FAdV/TaqMan probe/real-time fluorescence quantitative PCR引用本文复制引用
基金项目
国家自然科学基金青年基金(32102653)
河南省高等学校重点科研项目(23B230002)
河南省科技攻关计划(232102110083)
出版年
2024
NETL
NSTL
万方数据