应用套式PCR对恶性卡他热病毒在不同动物体内变异的研究

DETECTION OF MALIGNANT CATARRHAL FEVER VIRUS(MCFV) IN DIFFERENT ANIMAL SPECIES BY NESTED POLYMERASE CHAIN REACTION

应用本实验建立的三组套式ＰＣＲ（ＰＣＲ１、２、３）和一组以前报道的套式ＰＣＲ（ＰＣＲ４），对５９份外周血淋巴细胞（ＰＢＬ）ＤＮＡ样品进行了恶性卡他热病毒（Ｍａｌｉｇｎａｎｔｃａｔａｒｒｈａｌｆｅｖｅｒｖｉｒｕｓ，ＭＣＦＶ）核酸序列的检测。这些样品来自５１只羊，以及与羊接触而发病的６头牛和２只鹿。除ＰＣＲ４外，其它三组ＰＣＲ都能扩增现有４个角马型ＭＣＦＶ分离株。有６只羊在４组ＰＣＲ中都呈阴性，其余５３份样品经ＰＣＲ４检测均呈阳性。ＰＣＲ１只能从４５只羊体检出ＭＣＦＶＤＮＡ，未能从牛和鹿体检出病毒ＤＮＡ。ＰＣＲ２检测的所有样品均呈阴性。在ＰＣＲ３扩增中，除２头牛外，其它５１份样品均呈阳性。通过Ｓｏｕｔｈｅｒｎ杂交和限制性酶切分析，对ＰＣＲ１－４产物的特异性进行了鉴定。此外，敏感性实验表明，四组ＰＣＲ的差异也不明显。因此，本实验结果说明ＭＣＦＶ基因组在不同种动物之间发生了变异。

Three nested PCRs(designated as PCR1,2,and 3, respectively) were developed based on cDNA sequences of a US isolate(Minnesota) of malignant catarrhal fever virus(MCFV)and a 4.1 kb ORF of MCFV C500 strain.A previously established PCR(herein designated as PCR4) was also employed,in comparison with the three PCRs,to detect MCFV DNA from peripheral blood lymphocyte(PBL)samples of 45 MCFV-infected sheep,6 cattle and 2 deers with clinical MCF.Except PCR 4, each of other three PCRs amplified a fragment with desired size from four MCFV isolates,WC11,C500,Minnesota and Au-732.All 53 animals were detected positive by PCR 4.However,only 45 sheep showed positive reaction in PCR 1,and all 53 sheep,cattle,and deer samples were tested negative by PCR 2.In PCR 3, except for two cattle,all other 51 animals were reactive to DNA amplification.The specificities for PCR 1-4 were confirmed by Southern hybridization and restriction enzyme digestion.There was no apparent difference in sensitivity between the four sets of PCR.The results from the study indicated variation of MCFV genome in different animal species and may suggest that the variant(s) in sheep is a causative agent of MCF for other ruminants.