B亚型禽偏肺病毒逆转录套式PCR检测方法的建立与应用  

Development and application of a nested RT-PCR assay for detection of avian metapneumovirus subtype B

作　　者：薛聪[1] 刁有祥[1] 唐熠[1] 陈琳[1] 鞠小军[1] 崔京腾[1] 欧全宾[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2013年第2期171-174,180,共5页Chinese Journal of Veterinary Science

基　　金：山东省科技发展计划资助项目(2010GNC10912)

摘　　要：根据GenBank发布的B亚型禽偏肺病毒Fusion(F)基因的保守序列设计2对引物,建立了一种适用于B亚型禽偏肺病毒的逆转录套式PCR检测方法。采用该方法对B亚型禽偏肺病毒山东分离株进行检测,可以特异性地扩增出415bp的目的片段。此方法具有高度特异性和敏感性,以H9亚型禽流感病毒、新城疫病毒、传染性支气管炎病毒、传染性喉气管炎病毒作为模板进行扩增,结果均为阴性。经检测,该方法第1次PCR扩增的敏感性为107copies/μL,第2次扩增的敏感性为102copies/μL,第2次扩增敏感性比第1次高105倍。应用本方法对采自山东省不同地区的64份可疑临床病料进行检测,最终从64份疑似病料中检测出37份为阳性。结果表明,所建立的套式PCR检测方法具有特异、敏感、实用等优点,为B亚型禽偏肺病毒的快速诊断以及深入研究奠定了基础。To develop a nested RT-PCR method for the rapid detection of avian metapneumovirus in the clinical tests. According to the sequence of F gene of avian metapneumovirus published in Gen- Bank,two pairs of primers were designed and synthesized. A specific 415 bp fragment was amplified from RNA templates of avian metapneumovirus of Shandong isolates, but the amplification results of avian influenza virus （AIV） subtype Hg, Newcastle disease virus （NDV）, infectious bronchitis virus（IBV）, infectious laryngotracheitis virus（ILTV） were negative. Sensitivity of the ampli- fications by the nested RT-PCR assay in two times of detection was 1 × 10^7copies/μL and 1 × 10^2 eopies/μL,respectively. The sensitivity of the second amplifications was increased by 10S times and it had a higher detection rate of clinical samples. It was successfully applied to the clinical tests, which demonstrated it was more sensitive than the ordinary PCR and able to adjust the pseudo- negative results of the conventional PCR.

关 键 词：B亚型禽偏肺病毒 F基因 逆转录套式PCR 

分 类 号：S825.65[农业科学—畜牧学]