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

作　　者：刘琳[1] 宋亚鹏[1] 刘明洋 高文明[1] 陶明月 宋晓楠 张宜娜[1] 李新生[1] LIU Lin;SONG Yapeng;LIU Mingyang;GAO Wenming;TAO Mingyue;SONG Xiaonan;Zhang Yina;LI Xinsheng(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046

出　　处：《中国兽医学报》2024年第5期881-886,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金青年基金资助项目(32102653);河南省高等学校重点科研资助项目(23B230002);河南省科技攻关基金资助项目(232102110083)。

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。To detect serotype 1 FAdV infection in chickens,primers and TaqMan probes were designed according to the conserved 52K gene of serotype 1 FAdV in this study.Real-time fluorescence quantitative PCR optimized by the matrix method was used to detect the virus shedding of oral 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 R~2=0.996,indicating a good linear relationship.The results of the SPF chicken challenge showed that FAdV serotype 1 could be detected in the oral and cloacal swabs until 14 days post-infection,and the virus could also be detected 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PCR 

分 类 号：S852.65[农业科学—基础兽医学]