鸭甲肝病毒3型荧光定量PCR检测方法的建立及其在雏鸭体内分布规律  

Development of a real-time quantitative PCR for detection and investigation of in vivo dynamic distribution of duck hepatitis virus type 3

作　　者：林少莉 杨蕾[1] 张瑞华[1] 陈君豪[1] 王鑫[1] 夏琳琳[1] 谢之景[1] 姜世金[1] 

机构地区：[1]山东农业大学动物医学学院山东省动物生物工程与疾病防治重点实验室,山东泰安271018

出　　处：《中国兽医学报》2016年第5期723-727,733,共6页Chinese Journal of Veterinary Science

基　　金：山东省科技发展计划项目(2010GNC10914);山东省现代农业产业技术体系家禽创新团队基金资助项目(SDAIT-13-011-15)

摘　　要：为研究鸭甲肝病毒3型（DHAV-3）在雏鸭体内的动态分布情况,本研究建立了检测DHAV-3的Taqman荧光定量PCR方法,该方法在107-102范围内有良好的线性关系,相关系数0.997,检测下限为22copies/PCR。将200只2日龄雏鸭随机分为4组,依次对每组雏鸭肌肉注射0.2ELD50、2ELD50、20ELD50的DHAV-3（SD1201株）和生理盐水,并于感染后1、2、6、12、18、24、36、48、72、96h从各组分别随机取3只雏鸭,应用荧光定量PCR方法对其心脏、肝脏、脾脏、肾脏、胸腺、法氏囊的病毒含量进行检测。结果表明,DHAV-3于感染后1h即可在3个试验组雏鸭肝脏中检测到,病毒含量为102-103 copies/g,2h后在心脏、脾脏、肾脏、胸腺和法氏囊中均检测到该病毒。各器官中的病毒含量于感染后36-48h达到高峰期,至72h病毒含量仍维持稳定。此外,在整个感染过程中,被检器官中的病毒含量与初始感染剂量呈正相关。本研究结果有助于阐明DHAV-3的致病机理。To investigate in vivo dynamic distribution of duck hepatitis virus type 3（DHAV-3）,a real-time PCR assay for quantitative detection of DHAV-3 was established in this study.The standard curve of the method was generated with a range from 107 to 102 gene copies per reaction with 0.997 linear correlation value,and the minimum detection limit was 22 copies of DHAV-3cDNA per reaction.Two hundred 2-day-old ducklings were divided into 4groups randomly and the ducklings in each group were injected 0.2ELD50,2ELD50,20ELD50 of DHAV-3SD1201 strain and physiological saline.At 1,2,6,12,18,24,36,48,72 and 96hours post infection（hpi）,three ducklings in each group were randomly selected to collect heart,liver,spleen,kidney,thymus,bursa of Fabricius（BF）samples for viral detection.The result showed that DHAV-3could be detected at 1hpi in the livers of the three experimental groups and the viral titer was about 102-103copies/g.At 2hpi,DHAV-3could be detected in heart,spleen,kidney,thymus and BF.High levels of viral titers were detected in all inspected organs from 36-48 hpi and maintained stable until72 hpi.Besides,during the whole experiment period,the viral titers in inspected organs were proportional to the initial inoculated doses.The finding was helpful to elucidate the pathogenesis of DHAV-3.

关 键 词：鸭甲肝病毒3型 动态分布 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]