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首页> 外文期刊>Veterinarni medicina >Simultaneous detection of porcine pseudorabies virus, porcine parvovirus and porcine circovirus type 2 by multiplex real-time PCR and amplicon melting curve analysis using SYBR Green I
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Simultaneous detection of porcine pseudorabies virus, porcine parvovirus and porcine circovirus type 2 by multiplex real-time PCR and amplicon melting curve analysis using SYBR Green I

机译：使用SYBR Green I通过多重实时PCR和扩增子熔解曲线分析同时检测猪伪狂犬病病毒，猪细小病毒和2型猪圆环病毒

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Porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 can cause reproductive failure in pigs, and swine are often simultaneously infected by combinations of the three viruses. We here report the development of a SYBR Green I-based multiplex real time PCR assay for simultaneous detection of porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2. Three pairs of specific primers were designed for the porcine parvovirus-VP2, porcine pseudorabies virus-gH and porcine circovirus type 2-ORF2 genes. Viral genomes were identified based on their distinctive melting temperatures in singleplex PCR reactions. The melting temperature was 74.5 °C for the 313 bp amplicon of porcine parvovirus-VP2 gene, 87.5 °C for the 355 bp amplicon of porcine pseudorabies virus-gH gene and 80.5 °C for the 171 bp amplicon of the porcine circovirus type 2-ORF2 gene, respectively. The detection limit of the method ranged from 0.01–0.03 TCIDSUB 50 /SUB/ml for the three viruses. In addition, porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 viral loads were measured in 100 field samples, and the result showed that the concordance between real-time PCR and conventional PCR was 60.42%. The sensitivity and specificity of real-time PCR were 100% and 100%, while those of conventional PCR were 40.83% and 72.22%, respectively.
机译：猪细小病毒，猪伪狂犬病病毒和2型猪圆环病毒可引起猪的生殖衰竭，并且猪经常同时感染三种病毒。我们在此报告了基于SYBR Green I的多重实时PCR检测技术的发展，用于同时检测猪细小病毒，猪伪狂犬病病毒和2型猪圆环病毒。为猪细小病毒-VP2，猪伪狂犬病病毒设计了三对特异性引物-gH和猪圆环病毒2型ORF2基因。根据在单重PCR反应中独特的解链温度鉴定病毒基因组。猪圆环病毒2型猪细小病毒VP2基因的313 bp扩增子的解链温度为74.5°C，猪伪狂犬病毒-gH基因的355 bp扩增子的解链温度为87.5°C，171 bp扩增子的解链温度为80.5°C。 ORF2基因分别。该方法对三种病毒的检测极限范围为0.01–0.03 TCID 50 / ml。另外，在100个田间样品中检测到猪细小病毒，猪伪狂犬病病毒和猪圆环病毒2型病毒载量，结果显示实时PCR与常规PCR的一致性为60.42％。实时荧光定量PCR的敏感性和特异性分别为100％和100％，而常规PCR的敏感性和特异性分别为40.83％和72.22％。

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