荧光定量RT-PCR鉴别IBDV超强毒株与经典疫苗毒株  

Differentiation of Infectious Bursal Disease Virus between Classical and Very Virulent Strains by Real-Time RT-PCR

作　　者：汪振兴[1] 李树清[2] 孙建和[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]上海出入境检验检疫局,上海200135

出　　处：《上海交通大学学报（农业科学版）》2009年第2期120-124,共5页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：上海市科委技术标准专项(No.06DZ05133)

摘　　要：鸡传染性法氏囊病病毒(IBDV)超强毒株的出现给该病的防控带来了新的困难,有效鉴别IBDV超强毒株对IBD的防控有着积极意义。根据IBDV VP4基因的保守序列,设计了一对通用引物RPa/RPb,特异性扩增IBDV VP4基因720bp的目的片段。根据超强毒株和经典毒株VP4基因序列的差异,分别合成了FAM标记的超强毒荧光探针和JOE标记的经典毒荧光探针,建立了实时荧光RT-PCR鉴别IBDV超强毒株与经典疫苗毒株的方法。检测超强毒和经典毒的敏感性分别可达420和320个拷贝数。建立的实时荧光RT-PCR方法敏感性高、特异性强,可用于临床样品中IBDV超强毒和经典毒的鉴别诊断。Very virulent infectious bursal disease virus （vvIBDV） has lead to significant losses in the production of birds. To develop a rapid and efficient differential diagnostic assay is vital for the effective control of vvIBDV. A pair of consensus primers RPa/RPb based on the conserved sequence encoding VP4 was designed for the general diagnosis of IBD and a 720 bp segment can be amplified. A real-time RT-PCR assay was developed utilizing dual-labeled （JOE and FAM, respectively） fluorescent probes binding to the 720 bp segment that are specific to the classical or very virulent strains of IBDV. The classical sequence-specific probe demonstrated high sensitivity and specificity did not react with very virulent strains. The very virulent sequence-specific probe detected positively the SH95 vvIBDV isolate and no cross reaction was detected. This assay could be used to differentiate virus strains between vvIBDV and cIBDV with high sensitivity and specificity.

关 键 词：鸡传染性法氏囊病病毒(IBDV) Real-timeRT-PCR 鉴别诊断 荧光探针 

分 类 号：S851.347.201[农业科学—预防兽医学]