猪非典型性瘟病毒与猪流行性腹泻病毒双重PCR方法的建立与应用

Establishment and application of double PCR method between atypical porcine pestivirus and porcine epidemic diarrhea virus

为了快速准确地检测出猪非典型性瘟病毒(APPV)与猪流行性腹泻病毒(PEDV),根据Gen Bank中发布的PEDV S2基因和APPV NS3基因的序列,分别选取PEDV S2基因和APPV NS3基因的保守序列,设计、合成了1对特异性引物。通过对体系进行优化,分别扩增出190 bp和500 bp的特异性片段。建立的双重RT-PCR方法对猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪轮状病毒(RV)的检测均为阴性。对PEDV和APPV的最低检出量分别为1μl 2.65×10~5和1μl 3.56×10~4,对四川遂宁、眉山等地采集到的腹泻、肌肉震颤的病料进行检测,PEDV、APPV阳性病料检出率分别为41. 18%和11. 76%,经分别与特异性检测APPV和PEDV的单一RT-PCR法检测结果进行比较分析,符合率均为100%。与单一RT-PCR检测方法具有相同特异性和重复性,可用于临床检测。

In order to detect the atypical porcine pestivirus(APPV)and porcine epidemic diarrhea virus(PEDV)rapidly and accurately,according to the conservative sequences of PEDV S2 gene and APPV NS3 gene deposited in GenBank,a pair of specific primer was designed and synthesized.By optimizing the system,specific fragments of 190 bp and 500 bp were amplified,respectively.The detection results of porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV)and porcine rotavirus(RV)using this double RT-PCR method were negative.The minimum detectable amount of PEDV and APPV were 1μl 2.65×10 5 and 1μl 3.56×10 4,respectively.The detection rates for diarrhea and muscular tremor collected from Suining and Meishan in Sichuan province were 41.18%and 11.76%,respectively.The coincidence rates of the results detected by double RT-PCR method and the specific RT-PCR method for APPV and PEDV were all 100%.The double RT-PCR method has the same sensitivity,specificity and repeatability as the single RT-PCR assay and can be used for clinical detection.