摘要
根据猪瘟病毒(CSFV)以及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列特点,设计3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CSFV以及美洲型、欧洲型PRRSV的多重TaqMan荧光定量RT-PCR(qRT-PCR)方法。该方法具有特异性强、敏感性高、重复性好的特点,可以特异扩增CSFV和PRRSV,而与猪的其它常见病毒无交叉反应;对CSFV及美洲型、欧洲型PRRSV3种重组质粒标准品的检出下限均为2.68拷贝/μL;组内及组间重复试验的变异系数均小于1.5%。应用该方法对253份临床疑似样品进行检测,结果病原阳性106份,其中20份为CSFV阳性,86份为美洲型PRRSV阳性,11份为CSFV和美洲型PRRSV混合感染,未检测到欧洲型PRRSV。试验表明,研究建立的多重qRT-PCR方法可用于CSFV和PRRSV的快速鉴别检测及流行病学调查。
A multiplex quantitative TaqMan real-time RT-PCR （qRT-PCR） assay was established for differential detection of classical swine fever virus （CSFV） and North American （NA） and European （EU） genotype porcine reproductive and respiratory syndrome virus （PRRSV） under optimized reaction condition using the three pairs of specific primers and three TaqMan probes designed according to the different ge- nomic sequences among CSFV, NA-PRRSV and EU-PRRSV. The assay proved specifical, sensitive and re- producible, and helped amplify CSFV, NA-PRRSV and EU-PRRSV and meanwhile restraining from cross reaction with other important porcine pathogens. Detection limit of CSFV, NA-PRRSV and EU-PRRSV, all was 2.68 copies/μL ,respectively variable coefficient was less than 1.5 percent in both intra-and inter- assay. The established assay was used to detect CSFV and PRRSV in 106 of the 253 clinical samples detec- ted following the assay revealed positive, among which 20 samples were positive for CSFV, 86 samples for NA-PRRSV, 11 for both while none for EU-PRRSV. The results indicated that this assay could be used as an effective tool for rapid detection and epidemic surveillance of CSFV and PRRSV.
出处
《家畜生态学报》
北大核心
2015年第3期63-68,共6页
Journal of Domestic Animal Ecology
基金
广西科学基金项目(桂科青0832057)
广西科学研究与技术开发计划项目(桂科转14125004-22)
关键词
猪瘟病毒
猪繁殖与呼吸综合征病毒
多重TaqMan荧光定量RT-PCR
classical swine fever virus（PRRSV）
multiplex quantitative TaqMan（CSFV）
porcine reproductive andreal-time RT-PCR （multiplex qRTrespiratory syndrome virusPCR）
