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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Gen Bank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。
In order to improve the detection rate of transmissible gastroenteritis virus (TGEV) in colostrums and avoid the harm of virulent colostrums, a SYBR Green Ⅱ quantitative PCR (qPCR) method was established. According to the sequence of N gene of TGEV published by Gen Bank, a pair of specific primers were designed and synthesized. The N gene fragment of TGEV (174 bp) was amplified and a recombinant plasmid containing N gene fragment was constructed. The recombinant plasmid was used as The template established a specific detection of TGEV qPCR detection method. The sensitivity of the method was higher than that of the common PCR method. The qPCR method was used to detect 130 colostrums of sows collected from diarrhea pig farms in some areas of Sichuan Province from October 2016 to April 2017, PCR test methods for comparison. The results showed that 13 of 130 samples were positive for TGEV by qPCR, while only 5 samples of positive TGEV were detected by ordinary PCR. Indicating that the sensitivity of qPCR detection method is higher than the ordinary PCR method, more suitable for detection of porcine colostrum.