猪繁殖与呼吸综合征病毒cDNA探针的制备及初步应用

Production of PRRSV cDNA Probe Labeled with Digoxigenin

以猪繁殖与呼吸综合征 ( PRRS)病毒 ATCC VR-2 332株基因组为模板 ,通过 RT-PCR扩增出 586bp的 ORF6片段的 c DNA。将该片段克隆进 p GEM-T easy载体 ,转化大肠杆菌 JM1 0 9,成功获得重组质粒转化菌。将重组质粒经 Eco R 及 Sma 双酶切 ,回收到 1个 4 63bp的 c DNA片段 ,并制备出地高辛标记的c DNA探针。特异性检测表明 ,该探针对 PRRS病毒的 PCR产物呈现特异性 ,而对照的 HCV、PPV、PRV的核酸均呈阴性 ;敏感性检测表明 ,该探针对同源 DNA的检出限量为 4 pg。应用该探针对 6株 PRRS病毒北京分离毒及 ATCC VR-2 332株感染细胞进行原位杂交检测 ,结果均呈阳性。以上结果表明 。

A 586 bp cDNA fragment for ORF6 and part of ORF7 of PRRSV ATCC VR 2332 was amplified by RT PCR.The PCR product was cloned into pGEM T easy vector.A 463 bp fragment was obtained by EcoR Ⅰ/Sma Ⅰ digestion of recombinant plasmid and was labeled with digoxigenin dUTP.Dot blot hybridization for homogeneous plasmid DNA,PPV DNA,PRV DNA or HCV RNA was performed by using the probe.It was shown that homogeneous plasmid DNA was positive,but PPV DNA,PRV DNA and HCV RNA were negative.The probe could detect 4 pg homogeneous DNA.Cells infected with VR 2332 and six BJ isolates were examined by in situ hybridization and were positive.It was shown that the probe could be used to detect PRRSV in tissues by in situ hybridization.