摘要
根据GenBank中禽网状内皮组织增生病病毒(REV)基因组序列设计1对针对REVgp90基因的引物和1条特异性TaqMan探针,建立了一种快速检测REV病毒载量的TaqMan荧光定量PCR方法。通过对反应条件和反应体系的优化,使得该方法在1×101～1×108copies/μL范围内具有良好的线性关系,灵敏度达到10copies/μL,是常规PCR方法的100倍,该方法与其他禽病病毒无交叉反应,批间与批内重复性试验变异系数均小于2%。用REV HLJR0901株人工感染1日龄SPF雏鸡,定期剖检,对感染鸡体内各器官的病毒分布及载量进行检测,结果表明,在肝、脾、法氏囊、胸腺和腺胃中均可检测到病毒,其病毒载量并无明显差异。本研究结果表明,该方法具有良好的敏感性、特异性和重复性,可用于REV的定量检测,为该病毒的诊断及致病性的研究提供了技术手段。
A TaqMan real-time PCR assay with high specificity and sensitivity for rapid detection of reticuloendotheliosis virus（REV） was developed using a pair of primers and a TaqMan probe specific to the gp90 gene of REV.The assay was optimized to produce linearity from 1×10 1 copies/μL to 1×10 8 copies/μL in standard curve.There was no cross-reaction with other common avian viruses,and the assay had a detection limit of 10 copies of proviral DNA with a higher sensitivity than the conventional PCR methods.Furthermore,1 day-old SPF chickens were inoculated with the REV HLJR0901 strain and the virus loads were detected in multiple organs.The results showed that the virus could be detected in the liver,spleen,bursa,thymus and proventriculus and the virus loads in these organs showed no significant diffe-rence.The results suggested that the developed real-time quantitative PCR was a good tool for quantification of REV and would be useful for the detection and control of reticuloendotheliosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第11期1137-1141,共5页
Chinese Veterinary Science
基金
现代农业肉鸡产业技术体系建设项目(nycytx-42-G3-01)
