传染性法氏囊病病毒(IBDV)快速检测与分型技术

The Development of Rapid Diagnosis and Pathotyping Techniques for Infectious Bursal Disease Viruses

根据已发表的传染性法氏囊病病毒 (IBDV)核苷酸序列 ,在病毒结构蛋白 VP2编码基因的高变区两端外侧的保守序列内设计合成了 2条寡核苷酸引物 ,对各种不同致病性的 12株参考毒株进行了 RT- PCR检测。结果 :12个参考毒株均能扩增出约 6 79bp的目的片段 ,而对照的 5种鸡源性病原体 NDV、IBV、CAIV、E.coli、PM均未扩增到相应片段 ;对疑似 IBD的 34份临床病料进行检测 ,并同时在其扩增的片段内设计另 1对引物进行 Nested- PCR检测 ,结果基础 RT- PCR检测到 11份阳性 ,Nested- PCR检测到 2 3份阳性 ,后者的检出率大大提高。结果表明 ,建立的 RT- PCR诊断技术具有特异、快捷、敏感的特点。取限制性内切酶 Sac 和 Ssp 以及设计的 2条 vv IBDV(超强毒株 )特异性引物 ,分别应用 v VP2片段 PCR扩增产物的限制性内切酶分析和型特异性引物的 PCR扩增 2种方法 ,对 7个 IBDV分离参考毒株和 2 4个临床样品进行致病性分型。结果 ,确定 2 1株属于 c IBDV(经典毒株 ) ,8株属于 vv IBDV,2株未能确定 ;2种分型方法的结果基本一致 ,均可用于 IBDV的快速分型 ,型特异性引物的

Two sets of primers(Pta and Pts,IBDa and IBDs),flanking the hyper-variable region of VP2 gene(vVP2),were designed to run a reverse transcription polymerase chain reaction(primary RT-PCR) and Nested-PCR for the detection of IBDVs.Both of these assays can detect 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 primary RT-PCR and Nested-PCR.11 samples were positive by the primary RT-PCR,while 23 samples were positive by Nested-PCR that significantly increased the sensitivity of the detection.Restriction endonuclease analysis(REA) of vVP2 PCR products with SacⅠ and SspⅠ respectively and PCR using vvIBDV-specific primers were used to pathotype 7 reference and 24 field viruses.21 and 8 of the tested samples were classified as cIBDV and vvIBDV respectively by the identical results of REA and the primers directed-PCR,while 2 samples were undetermined.The results demonstrated that both of the techniques can be used for the rapid pathotyping of IBDV,and the latter has the advantages on the simplicity and rapidity.And the results also reveal the prevalent pathotypes dominating in Guangxi chickens during the years of 1993 to 2003.