猪传染性胃肠炎病毒SYBR GreenⅡ荧光定量PCR检测方法的建立及在初乳检测上的应用

摘要

为了提高初乳中猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)的检出率,避免初生仔猪受到带毒初乳的危害,建立了一种SYBR GreenⅡ荧光定量PCR(qPCR)检测方法.根据GenBank已发表的TGEV的N基因序列,选择保守区域设计、合成了1对特异性引物,扩增TGEV的N基因片段(174 bp),构建含有N基因片段的重组质粒,以重组质粒作为模板建立了特异性检测TGEV的qPCR检测方法.该方法的灵敏性比普通PCR方法高,应用建立的qPCR检测方法,对2016年10月-2017年4月采集自四川省部分地区腹泻猪场的母猪初乳130份进行检测,并与普通PCR检测方法进行比较.结果显示:用qPCR方法检测,130份样本中有13份为TGEV阳性,而普通PCR方法只检测到5份TGEV阳性样本.说明qPCR检测方法的敏感性高于普通PCR方法,更适合用于猪初乳检测.%A SYBR Green II fluorescence quantitative PCR ( qPCR) method was established to improve the detec-tion rate of transmissible gastroenteritis virus ( TGEV) in colostrum, and to avoid the damage to primary piglets by toxic co-lostrum. According to the N gene sequence of TGEV published in GenBank, the conservative area was selected to design a pair of specific primers, and the amplified N gene fragment was 174 bp. A recombinant plasmid contai-ning N gene fragment was used as template to establish a qPCR method for the specific detection of TGEV. The qPCR method was used to detect 130 samples of sow colos-trum collected from October 2016 to April 2017 in some parts of Sichuan province ( diarrhea pig farm) . The results showed that 13 of the 130 samples were positive for TGEV by qPCR, and only five samples of TGEV were detected by common PCR. This indicated that qPCR method was more sensitive than the common PCR method, and the qPCR method was more suitable for colostrum testing.