美洲型与欧洲型PRRSV多重TaqMan荧光定量RT-PCR鉴别检测方法的建立及应用

Establishment and application of a multiplex TaqMan real time RT-PCR assay for simultaneous and differential detection of North American and European genotype PRRSV

根据猪繁殖与呼吸综合征病毒(PRRSV)美洲型经典毒株、高致病性变异毒株以及欧洲型毒株基因组的序列差异,设计2对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能同时检测并区分PRRSV美洲型经典毒株、变异毒株以及欧洲型毒株的多重荧光定量RT-PCR方法。该方法线性关系好,标准曲线的相关系数均达到0.999以上;特异性强,只有PRRSV出现阳性反应,而与口蹄疫病毒、猪瘟病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型均无交叉反应;敏感性高,经典毒株、变异毒株、欧洲型毒株的检出下限分别为1.13、1.37、1.39copies/μL,均比普通RT-PCR敏感100倍;重复性好,组内及组间变异系数均小于1.5%。应用所建立的方法对采集的282份临床病料进行检测,结果 PRRSV阳性101份,其中95份为变异毒株、6份为经典毒株、4份为变异毒株和经典毒株混合感染,未检测到欧洲型毒株。结果表明,本研究建立的多重TaqMan荧光定量RT-PCR方法可为PRRSV的快速鉴别诊断及流行病学调查提供有效的技术手段。

A multiplex TaqMan real time RT-PCR assay was established for differential detection of the classical(C)and highly pathogenic(HP)North American(NA)genotype and the European(EU)genotype porcine reproductive and respiratory syndrome virus(PRRSV).The assay utilized two pairs of specific primers and three TaqMan probes which were designed according to the different genomic sequences among C-PRRSV,HP-PRRSV and EU-PRRSV.The assay was highly specific,sensitive and reproducible.The correlation coefficient of the standard curves were over 0.999.It could amplified only from PRRSV,but not from other important porcine pathogens such as foot-and-mouth disease virus,classical swine fever virus, pseudorabies virus,porcine parvovirus,porcine circovirus type 2 and so on.The detection limit of C-PRRSV,HP-PRRSV and EU-PRRSV was 1.13,1.37and 1.39copies/μL,respectively,which was 100 times higher than that of their corresponding conventional RT-PCR.The coefficient of variation was less than 1.5percent for both intra-assay and inter-assay.The established assay was used to detect PRRSV in 282clinical samples and 101samples were positive for PRRSV,of which 95samples were positive for HPPRRSV,6samples for C-PRRSV and 4samples for both HP-PRRSV and C-PRRSV,but no samples for EU-PRRSV.The results indicated that this assay could be used as an effective tool for rapid detection and epidemiological surveillance of PRRSV.