猪捷申病毒TaqMan实时定量RT-PCR方法的建立

Development of TaqMan real-time RT-PCR assay for detection of porcine teschovirus

为建立猪捷申病毒(PTV)的早期检测及定量分析方法,本研究基于PTV 11个血清型基因组5'端非编码区保守序列,设计引物和TaqMan探针,建立了检测PTV的TaqMan实时定量RT-PCR方法。应用该方法对PTV、猪细小病毒、猪繁殖与呼吸综合症病毒、猪圆环病毒2型、猪伪狂犬病毒以及猪瘟病毒进行特异性试验,结果除PTV为阳性外其它均为阴性;针对PTV最低可检测到10个拷贝;批内、批间重复试验的变异系数均小于3%。应用建立的方法与病毒分离方法分别对91份临床样品进行检测,检出率分别为79.12%和57.14%,两者的符合率是78.02%。经临床应用表明,该实时定量RT-PCR方法可为PTV的早期诊断及定量分析提供技术手段。

To detect the porcine teschovirus （PTV）, a real-time RT-PCR based on TaqMan was established with a pair of primers and a TaqMan probe targeting the highly conserved sequences of the 5＇-untranslated region of 1 to 11 serotypes of PTV. The results indicated that the real-time RT-PCR was specific for detection of PTV with a detection limit of 10 copies, but not for porcine parvovirus, porcine circovirus 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and classical swine fever virus. The coefficient of variation of inter-assay and intra-assay were less than 3%. A total of 91 clinical samples were tested by the real-time RT-PCR comparing with virus isolation, and positive rates were 79.12% （72/91） and 57.14% （48/91）, respectively. In conclusion, the developed real-time RT-PCR assay was an effective method for detection and quantification of PTV in organs of infected pigs.