猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪博卡病毒多重PCR检测方法的建立

Establishment of Multiplex PCR for Detection of TGEV , PEDV and PBoV

根据GenBank登录的猪传染性胃肠炎病毒(TGEV)S基因序列、猪流行性腹泻病毒(PEDV)M基因和猪博卡病毒(PBoV)NP1基因序列,设计合成3对引物。通过优化反应条件,对TGEV、PEDV RNA反转录获得的cDNA模板和PBoV的DNA模板进行多重PCR扩增,同时得到3条与试验设计相符的1 060bp(TGEV)、462bp(PEDV)及258bp(PBoV)特异性条带,建立了同时检测TGEV、PEDV和PBoV的多重PCR方法。结果表明,在同一PCR反应体系中,可以同时检测这3种病毒核酸片段,而对其他病毒的检测均为阴性;敏感性分析表明,该方法的最低检测限分别为35、20、10pg的核酸。采用该方法对60份临床样品进行检测,TEGV阳性率为1.6%,PEDV阳性率为6.7%,PBoV阳性率为21.7%,表明建立的方法可以应用于临床检测。

To develop a method for simultaneous detection of transmissible gastroenteritis virus （TGEV）, porcine epidemic diarrhea （PEDV） and procine boeavirus（PBoV）,a multiplex PCR was established with 3 pairs of specific primers designed based on the conserved sequences in S gene of TGEV,M gene of PEDV and NP1 gene of PBoV. Three special fragments of 1 060 bp （TGEV） ,462 bp （PEDV） and 258 bp（PBoV） were amplified by the optimized multiplex PCR and no amplification was achieved from other negative con- trol groups. The sensitivity test showed that the PCR could detect 35 pg of TGEV RNA, 20 pg of PEDV RNA and 10 pg of PBoV DNA. 60 clinic samples of sick pigs were detected by the multiplex PCR and the detection rate was 1.6% （1/60） for TGEV,6.7% （4/60） for PEDV and 21.7% （13/60） for PBoV, re- spectively,which indicated that the multiplex PCR for detecting TGEV, PEDV and PBoV was rapid, spe- cific and sensitive, and could be used in clinic detection.