牛奶中志贺氏菌PCR检测方法的建立

Development of detection method for shigella in milk by polymerase chain reaction

根据Genbank志贺氏菌侵袭性质粒抗原H(ipaH)基因序列,自行设计引物,扩增特异的326bp核酸片段,经过优化PCR扩增条件,建立了志贺氏菌特异、敏感、快速的PCR检测方法,并对牛奶中的志贺氏菌进行了检测。特异性试验结果表明,志贺氏菌参考菌株均能扩增出特异的核酸片段,大肠杆菌、巴氏杆菌、金黄色葡萄球菌、沙门氏菌、蜡样芽孢杆菌、变形杆菌、绿脓杆菌的扩增结果均为阴性。敏感性试验结果表明,采用试剂盒提取基因组,该方法的敏感性可达到1.75×102cfu/mL。人工污染牛奶的模拟检测结果表明,PCR方法的检测限为1.75×103cfu/mL。

According to Genbank shigella invasion plasmid antigen H(ipaH)gene sequences,a pair of primers were designed for amplification of specific nucleic acid fragment of 326 bp,after optimizing the PCR amplification conditions,established a Shigella-specific,sensitive and rapid PCR detection method.Specificity test results showed that able to amplify the specific nucleic acid fragment,E.coli,Pasteurella multocida,Staphylococcus aureus,Salmonella,Bacillus cereus,Proteus mirabilis,Pseudomonas aeruginosa were negative.Sensitivity test results showed that the sensitivity of the method can reach 1.75×102 cfu/mL.The detection limits of this method for artificially contaminated milk was 1.75×103 cfu/mL.