猪流感病毒和猪繁殖与呼吸综合征病毒同步检测和鉴别诊断方法的建立

Establishment of multiplex real-time RT-PCR for simultaneous detection and differentiation of SIV and PRRSV

为了准确鉴别猪流感病毒(SIV)和猪繁殖与呼吸综合征病毒(PRRSV)的感染,本研究建立了可同时检测和鉴别SIV、NA-PRRSV和EU-PRRSV的一步法多重荧光RT-PCR方法,并进行了临床应用。研究结果表明,所建立的多重荧光RT-PCR最低检测限可达1~10 copies/?L,特异性为100%。SIV、NA-PRRSV和EU-PRRSV单荧光RT-PCR与多重荧光RT-PCR相比,相关性系数R2值大于0.999,所有扩增效率E值均在90%~100%之间,扩增性能良好,多重混合对特异性和敏感性未产生明显干扰。对病毒核酸样品的验证100%符合,从89份NA-PRRSV阳性样品中检测出NA-PRRSV阳性88份(88/89),从28份SIV阳性样品中检测出SIV阳性28份(28/28),从这两类阳性样品中检测出SIV和NA-PRRSV双阳性4份(4/117);从82份NA-PRRSV阴性样品中检测到EU-PRRSV和SIV阳性各1份;从105份未知感染状态的临床样品中筛查出2份SIV阳性和3份NA-PRRSV阳性。本研究建立的多重荧光RT-PCR方法实现了一次检测同时鉴别多种病毒的目的,适用于猪流感和猪繁殖与呼吸综合征混合感染的快速筛查。

To distinguish accurately between infections with swine influenza virus(SIV)and porcine respiratory and reproductive syndrome virus(PRRSV),one multiplex real-time RT-PCR to detect and differentiate simultaneously the infection of SIV and genotypes of North American PRRSV(NA-PRRSV)and European PRRSV(EU-PRRSV)is established and validated in this study.The results showed that all the real-time RT-PCR has good amplifications with amplification efficiencies(E)of 90%—100%,the correlation coefficients(R2)of over 0.999 and specificity of 100%,and the detection limit is 1—10 copies/?L.No significant difference is observed between any single real-time RT-PCR and multiplex ones,which indicates multiplexing the three targets into one reaction did not cause any interruption on specificity,sensitivity and correlations.Method validation tests showed that the newly developed multiplex real-time RT-PCR produced the expected results,with 88 samples were test positive for NA-PRRSV from known 89 positive NA-PRRSV samples,28 samples were test positive for SIV from known 28 positive SIV samples,and 4 samples were test positive for both NA-PRRSV and SIV among them.1 sample was test positive for EU-PRRSV and SIV,respectively,from known 82 negative NA-PRRSV samples.2 positive SIV and 3 positive NA-PRRSV were screened,respectively,from 105 unknown clinical samples.The method developed here can be used to rapidly screen the mix-infection with SIV and PRRSV,this provides a good alternative to detect and differentiate simultaneously co-infections of multi-pathogens based on one single reaction.