同时检测水禽3种常见病原菌的环介导间接PCR方法的建立

Development of loop-mediated indirect PCR for simultaneously detecting 3 pathogenic bacteria of waterfowl

为建立同时检测大肠杆菌、禽源多杀性巴氏杆菌和鸭疫里默氏菌等水禽常见病原菌的环介导间接PCR方法，根据大肠杆菌的gapA基因、禽源多杀性巴氏杆菌的KMTl基因、鸭疫里默氏菌的OmpA基因序列，分别设计引物、标记于猪线粒体基因片段的两端，制备不同细菌的捕获探针；将捕获探针与待检测菌的基因组杂交、补平缺口、环化。采用1对引物反向扩增捕获探针，建立检测水禽3种常见病原菌的环介导间接PCR方法，扩增片段大小分别为328，422和504bp。沙门菌、金黄色葡萄球菌、小鹅瘟病毒的检测结果为阴性。该方法能检测出100Pg的细菌基因组DNA，与常规PCR的符合性为]00％。该方法可用于水禽3种常见病原菌的同时检测，是一种特异性好、灵敏度高的快速病原学诊断方法。

The objective of this test was to establish ultaneously detecting Escherichia coli , Pasteurella a loop-mediated indirect PCR method for sim multocida and Riemerella anatipestifer. Ac cording to the conserved sequences of Escherichia coli gapA gene and Pasteurella multocida KMT1 gene,Riemerella anatipestifer OmpA gene, there specific probes were designed and labeled on both ends of the porcine mitochondrial gene fragments and then 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 bacteria species indirectly. The loop-mediated indirect PCR assay was developed,and the specific PCR products were 328 bp for Escherichia coli and 422 bp for Pasteurella multocida and 540 bp for Riemerella anatipestifer. The method did not amplified Salmonella, Staphylococcus aureus and goose parvovirus. The detection limits of this method was 100 pg. The results of the assay were in accordance with the results of conventional PCR. The loop-mediated indirect PCR assay developed in this study is a fast specific and sensitive method for detecting the 3 pathogenic bacteria of waterfowl at the same time.