同时检测鸭疫里默氏菌和大肠杆菌的环介导间接PCR方法的建立

Development of loop-mediated indirect PCR for simultaneously detecting Riemerella anatipestifer and Escherichia coli

为建立同时检测鸭疫里默氏菌和大肠杆菌的环介导间接PCR方法,本研究根据鸭疫里默氏菌外膜蛋白A基因和大肠杆菌的gapA基因的保守序列设计两对特异性探针,分别标记于猪线粒体基因片段的两端形成两种不同的捕获探针,将捕获探针与待检测菌的基因组杂交、补平缺口、环化后,采用1对引物反向扩增捕获探针,建立检测鸭疫里默氏菌和大肠杆菌的环介导间接PCR方法,扩增片段分别为540 bp和328 bp,本研究建立的方法对金黄色葡萄球菌、禽多杀性巴氏杆菌、沙门氏菌的检测结果为阴性;能够检测出10 pg的鸭疫里默氏菌和大肠杆菌的基因组DNA,与常规PCR的符合率为100%。本研究建立的环介导间接PCR方法是一种特异性好、灵敏度高的快速病原学诊断方法,为同时检测鸭疫里默氏菌和大肠杆菌提供了一种可行的方法。

To establish a loop-mediated indirect PCR method for simultaneously detecting Riemerella anatipestifer and Escherichia coli,two specific probes were designed according to the conserved sequences of R.anatipestifer outer membrane protein A gene and E.coli glyceraldehyde-3-phosphate dehydrogenase A gene,and labeled on both ends of the porcine mitochondrial gene fragments and then two different capture probes were formed.After hybridizing with the genome of bacteria,gap filling and cyclizing,the capture probes were amplified by reverse PCR with a pair of primers to detect the two bacteria species indirectly.The loop-mediated indirect PCR assay was developed,and the specific PCR products were 540 bp for R.anatipestifer and 328 bp for E.coli.The specificity of this method was high,and no amplification was achieved to Staphylococcus aureus,Pasteurella multocida and Salmonella.The detection limits of this method was 10 pg genome DNA of the R.anatipestifer and E.coli.The results of the loop-mediated indirect PCR assay was consistent with the results of conventional PCR by detection the 20 clinical samples.The loop-mediated indirect PCR assay developed in this study was a fast,specific and sensitive method for detecting R.anatipestifer and E.coli at the same time.