猪细小病毒分离、核酸探针研制及应用

ISOLATION OF PORCINE PARVOVIRUS AND DEVELOPMENT OF PPV DNA PROBES FOR THE DETECTION OF PPV

采用分子克隆技术制备了ＰＰＶ－ＤＮＡ－Ｃ片段（ＰＰＶ－ＲＦ－ＤＮＡ，经ＰｓｔＩ／ＨｉｎｄⅢ的双酶切片段，称为Ｃ片段）及含Ｃ片段的ＰＵＣ１９重组质粒（ＰＵＰ），利用地高辛（Ｄｉｇｏｘｉｅｎｉｎ）标记，分别制备探针２和探针１。对猪细小病毒ＤＮＡ进行斑点杂交，两种探针均为阳性，而对照的猪瘟病毒、猪伪狂犬病毒、乙型脑炎病毒及ＰＫ－１５细胞的核酸均为阴性。并且后一种探针的敏感性高于前者，两种探针的ＤＮＡ检出限量分别为４０ｐｇ和４ｐｇ。同时对７例疑是猪细小病毒自然感染病理材料，经过处理后直接点样检测，均为阳性反应，而对照的健康猪组织，猪瘟病料，猪伪狂犬病病料检测结果为阴性。从７例自然病例中分离７株病毒。通过血凝试验、血凝抑制试验、免疫电镜技术及聚合酶链式反应等试验对分离毒进行了鉴定，结果分离到病毒均为猪细小病毒。结果表明，两种探针均具有高度的特异性、敏感性。这种方法对猪细小病毒感染的诊断、流行病学调查及检疫是一种有效、可靠的方法。

A PPV DNA C fragment obtained by Pst I/Hind Ⅲ digestion and its recombinant plasmid,PUC19(PUP),were labelled with Digoxigenin as DNA probes for porcine Parvovirus(PPV).Dot blot hybridization with the two probes showed the positive result for PPV DNA,but negative for the nucleic acid samples obtained from Hog cholera virus,Pseudorabies virus,Japanese B Encephalitis virus and PK 15 cells.The sensitivity of PUP probe,DNA detection limit being 4pg,was superior to that of the PPV DNA C fragment probe.At the same time,the clinic samples(aborted foetuses)from seven naturally infected cases were treated and determined by the same method.All of the samples were positive,while the samples from healthy swine tissue,cases infected with swine fever virus,and cases infected with Pseudorabies virus were negtive. Seven strains of the virus were isolated from pig farms by the authors,These viruses were identified as PPV by hemagglutination and hemagglutination inhibition tests,by Immunoelectronic microscope technology,and by polymerase chain reaction. It was demonstrated that the two probes were of high speciality and senesitivity.It could be concluded that the method established here will be valid and reliable for the dignosis,epidemiological investigation and quarantine of PPV.