传染性法氏囊病快速诊断技术的建立及其应用

The development of rapid diagnostic technique for IBD

根据已发表的IBDV各致病型毒株的序列,在病毒结构蛋白VP2编码基因的高变区(VP2variabledo-main,vVP2)两端外侧的保守序列内设计合成了2条寡核苷酸引物,对各种不同致病型的12株参考毒株进行逆转录酶—聚合酶链式反应(RT-PCR)的检测.结果12个参考毒株均能扩增出约679bp的目的片段,而对作为阴性对照的常见5种鸡病病原NDV、IBV、CAIV、E.coli、pM均没有扩增出任何片段;应用建立的技术对疑似IBD的34份临床病料进行检测,并同时在基础RT-PCR扩增的片段内设计另一对引物进行Nested-PCR检测.结果基础RT-PCR方法检测到11份阳性,Nested-PCR检测到23份阳性.研究的结果表明建立的IBD的RT-PCR诊断技术具有特异、快捷、敏感的特点.在此基础上设计的Nested-PCR则大大提高了阳性检出率(提高52.1%).

Two sets of primers (Pta and Pts, IBDa and IBDs), flanking the hypervariable region of VP2 gene, were designed to run a reverse transcription polymerase chain reaction (primary RTPCR) and NestedPCR. Both of these assays could amplify all of 12 reference strains which including pathotypes cIBDV, vvIBDV and vIBDV, but not the 5 negative reference pathogens of chicken. 34 field samples, which were collected from different places of Guangxi during the years of 1993 to 2003, were detected by using the developed primary RTPCR and NestedPCR. 11 samples were positively detected by the primary RTPCR, while 23 samples were positively detected by the NestedPCR, significantly increasing the sensitivity of the technique. 