4种病原菌“单管一步法”PCR快速检测方法的建立  

Establishment of"Single-tube One-step"Rapid PCR Detection Method for Four Pathogens

作　　者：潘雅君 徐汪节[1] 彭丽娜[1] 乔中东[2] 王朝霞[1] PAN Yajun;XU Wangjie;PENG Lina;QIAO Zhongdong;WANG Zhaoxia(Laboratory Animal Center,Shanghai Jiao Tong University,Shanghai 200240,China;School of Life Science and Technology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学实验动物中心,上海200240 [2]上海交通大学生命科学技术学院,上海200240

出　　处：《实验室研究与探索》2021年第5期68-72,共5页Research and Exploration In Laboratory

基　　金：上海交通大学医工交叉项目(YG2017MS07)。

摘　　要：为了检测实验动物常见的4种条件性致病菌:肺炎克雷伯杆菌、金黄色葡萄球菌、嗜肺巴斯德杆菌和绿脓杆菌,提出一种可更准确、灵敏、快速、简便的多重巢式PCR方法(MN-PCR)。根据待检微生物16S rDNA序列的保守区和可变区分别设计通用引物和特异引物,通过两种引物长短差异化创新设计,将模板富集和特异片段扩增两个过程整合为单个反应体系内的一步PCR反应。结果表明,MN-PCR方法能够在标准株DNA混合模板中同时扩增出4条待检测片段;而在阴性对照组中未检测出任何片段。MN-PCR方法在实际检测应用中能够成功检测出细菌感染阳性样本,且未扩增出其他微生物的特异性条带。建立的MN-PCR体系可以同时检测实验动物咽拭子等临床样本中的多种病原菌,与常规多重PCR方法相比,该方法具有特异性和灵敏度高、操作简便、节省时间等优点。The purpose of this study is to establish a more accurate,sensitive,fast and simple method that can detect four common conditional pathogens(Klebsiella pneumoniae,Staphylococcus aureus,Rodentibacter pneumotropica and Psueudomonas aeruginosa)in laboratory animals.According to the conservative and variable regions of the 16 S rDNA sequence of the microorganisms to be tested,universal primers and specific primers are designed respectively.Through the innovative design of the length of the two primers,the two processes of template enrichment and specific fragment amplification are integrated into a single reaction system,i.e.,one-step PCR reaction.The results show that the MN-PCR method can simultaneously amplify four fragments to be detected in the standard strain DNA mixed template,but no fragments are detected in the negative control group.The MN-PCR method can successfully detect positive samples of bacterial infection in actual detection applications,and no specific bands of other microorganisms have been amplified.The established MN-PCR system can simultaneously detect multiple pathogens in clinical samples such as experimental animal throat swabs.Compared with conventional multiplex PCR methods,this method has the advantages of high specificity and sensitivity,simple operation,and time saving.

关 键 词：多重巢式PCR 肺炎克雷伯杆菌 金黄色葡萄球菌 嗜肺巴斯德杆菌 绿脓杆菌 小鼠 

分 类 号：Q93-33[生物学—微生物学]