一步法多重RT-PCR检测猪圆环病毒2型和猪繁殖与呼吸道综合征病毒

Simultaneous detection of porcine circovirus type 2 and porcine respiratory and reproductive syndrome virus by one-step multiplex RT-PCR

根据猪圆环病毒2型(PCV2)的ORF2和猪繁殖与呼吸道综合征病毒(PRRSV)的ORF7相对保守区序列分别设计了两对引物PH1/PH2和PT1/PT2,将PCV2和PRRSV细胞毒按1:1混合,设为模拟样品,采用病毒基因组DNA/RNA提取试剂盒同时提取PCV2DNA和PRRSV RNA核酸,利用2对引物进行一步法多重RT-PCR。结果同时得到与实验设计相符的560bp(PCV2)和398bp(PRRSV)特异性扩增条带,而对其他5种猪病原的扩增均为阴性。敏感性试验表明,建立的一步法多重RT-PCR方法可检测出10ngPCV2DNA和5ngPRRSV RNA。

An one-step Multiplex RT-PCR assay was developed using two pairs of primers （PHI/PH2 and PTI/PT2） designed according to the conservative sequences of porcine circovirus type 2 （PCV2） ORF2 and porcine respiratory and reproductive syndrome virus （PRRSV） ORF5 respectively. PCV2 DNA and PRRSV RNA was extracted from the mixed sample of PCV2 and PRRSV and used as PCR template. Two specific PCR fragments of PCV2 560 bp and PRRSV 398 bp were amplified. No PCR products were detected from other four porcine pathogenic viruses. The assay has a sensitivity of detecting 10 ng of PCV2 DNA and 5 ng PRRSV RNA.