高致病性猪生殖与呼吸综合征病毒NASBA-ELISA检测方法的建立

Development of an NASBA-ELISA for detection of highly pathogenic porcine reproductive and respiratory syndrome virus

针对猪生殖与呼吸综合征病毒(PRRSV)ORF1中编码Nsp2的基因序列设计引物,建立了核酸序列依赖性扩增(NASBA)方法,并结合微孔板中液相杂交和酶标显色反应检测NASBA扩增产物,建立了检测高致病性PRRSV的NASBA-ELISA。琼脂糖凝胶电泳显示,NASBA方法扩增出了特异性的目的条带。将NASBA产物RNA系列稀释后同时进行了ELISA和Northern杂交。结果显示,ELISA和Nor-thern杂交最高可分别检测到1∶50和1∶30稀释的产物RNA。采用建立的NASBA-ELISA可检测到101.83TCID50/mL的HuN4株PRRSV。该方法只对3株高致病性PRRSV的检测结果为阳性,对经典株PRRSV和其他猪源病毒的检测结果均为阴性。结果表明,NASBA-ELISA能够敏感并特异地检测高致病性PRRSV。

A method to detect highly pathogenic porcine reproductive and respiratory syndrome virus （PRRSV） was developed based on nucleic acid sequence-based amplification（NASBA） with primers targeting the gene ORF1 encoding Nsp2, followed by a liquid phase hybridization in microtiter plates and colorimetric detection of the NASBA products. Gel electrophoresis revealed that a distinct band was amplified. The RNA products by NASBA were diluted and then detected by ELISA and Northern-blot. ELISA and Northern-blot could detect the products diluted at 1： 50 and 1 ： 30,respectively. The developed NASBA- ELISA could detect as little as 10183 TCID50/mL of HuN4 PRRSV. The three isolates of highly pathogenic PRRSV could be detected,but the isolates of classical PRRSV and other swine viruses could not be detected by this method. The results showed that the NASBA- ELISA can detect highly pathogenic PRRSV sensitively and specifically.