地高辛标记PPV-DNA-C及PUP重组质粒探针的比较研究

Study on Comparison of the PPV DNA C Probe and PUP Recombinant Plasmid Probe Labelled with Digoxigenin

应用分子克隆技术制备的猪细小病毒复制型ＤＮＡ的ＰｓｔⅠ／ＨｉｎｄⅢ双酶切片段Ｃ及被克隆到ＰＵＣ１９中的Ｃ片段形成的重组质粒ＰＵＰ利用非放射性的地高辛标记后制备两种探针。分别对不同来源的猪细小病毒ＤＮＡ及ＰＵＰ重组质粒于硝酸纤维素膜上打点杂交，免疫呈色后均为阳性反应，而对照的猪瘟病毒、乙型脑炎病毒、伪狂犬病毒、ＰＫ－１５细胞的核酸均为阴性反应。通过对已知量的ＰＰＶ－ＤＮＡ检测发现Ｃ探针及ＰＵＰ探针的最低检出限量分别为４０ｐｇＤＮＡ和４ｐｇＤＮＡ。

A PPV DNA C fragment obtainted by PstⅠ/Hind Ⅲ digestion and PUP recombinant plasmid formed by C fragment which was cloned to PUC 19 ,were labelled with nonradioactive Digoxigenin as DNA probes for Porcine Parvovirus(PPV) via molecule clone technology.Dot blot hybridization with the two probes on Nitrocellulose Membranes showed the positive results for PPV DNA from different resources and PUP recombinant plasmid,but negative for the nucleic acid samples obtained from a series of control virus.The lowest DNA detection limits of the PPV DNA C fragmentprobe and PUP probe were 40pg and 4pg respetively on detection of the PPU DNA known its quantity.The sensitivity of the recombinant plasmid PUP probe was ten times of the PPV DNA C fragment probe.