牛乳中沙门氏菌的荧光定量PCR检测方法的建立

Establishment of fluorogenic quantitative PCR method for detection of Salmonella in milk

为建立一种牛乳中沙门氏菌的荧光定量PCR快速检测方法,根据沙门氏菌invA基因序列和荧光定量PCR要求设计了合适的特异性引物,然后提取菌体DNA,对目的基因进行克隆,以重组质粒为模板进行荧光定量PCR扩增,绘制标准曲线,以沙门氏菌DNA为模板进行荧光定量PCR扩增,确定其扩增条件。结果表明,建立的方法特异性强,与志贺氏菌等致病菌无交叉反应,且方法的灵敏度高(为0.1 ng/L)。以掺入沙门氏菌的牛乳为样品,可检测到牛乳中44.2 mL^(-1)的沙门氏菌。该方法适用于快速、准确检测牛乳中的沙门氏菌,为实验室快速检测致病菌提供参考。

In order to establish a fast fluorogenic quantitative PCR detecting method of Salmonella in milk, the suitable and specific primers were designed according to the invA gene sequence of Salmonella and requirement of fluorogenic quantitative PCR. Then the bacteria DNA was extracted and the target gene was cloned. We used fluorogenic quantitative PCR method to amplify the template of recombinant plasmid and draw standard curve, meanwhile, using fluorogenic quantitative PCR method to amplify DNA template of Salmonella and confirm the amplified conditions. The specificity of established method is high and no cross reactions with pathogenic bacteria, such as shigella, the detected sensitivity reached 0.1 ng/L. The method can detect 44.2 mL-1 samples of Salmonella in milk. Therefore, the method is suitable for fast and accurate detecting salmonella in milk and provide reference for rapid detection of pathogenic bacteria in laboratory.