鸭疫里默菌环介导等温扩增检测方法的建立

Detection of Riemerella anatipestifer by loop-mediated isothermal amplification

采用对应于鸭疫里默菌16SrRNA基因442~461bp序列的5条特异引物,建立了环介导等温扩增(LAMP)反应体系,加入试剂盒提取的鸭疫里默菌DNA后,置63℃反应60min,通过监测反应液浊度判断结果。在此基础上,用煮沸10min的方法代替试剂盒提取DNA方法,并用显色方法代替浊度检测和琼脂糖凝胶电泳方法判断结果,通过敏感性、特异性、病原消长规律试验验证方法的准确性。另外,对临床采集的135只患病鸭的心血、脑组织、肝脏和心脏共540份样品提取DNA后,用LAMP和PCR进行检测,同时进行细菌分离和鉴定,比较各种方法检测结果的符合率。结果显示,菌液提取DNA后,LAMP和PCR方法最低分别可检测到2和10个CFU的鸭疫里默菌。菌液煮沸代替试剂盒提取DNA方法,使LAMP的敏感性降低5倍。显色法、浊度检测和琼脂糖凝胶电泳方法判断结果的敏感性和特异性相同。细菌分离法共获得201株鸭疫里默菌,且经LAMP与PCR法检测均呈阳性,LAMP与PCR法检出阳性样本总数分别为271和254份,LAMP方法阳性检出率高于PCR方法。建立的鸭疫里默菌LAMP检测方法具有较高的敏感性,进一步用菌液煮沸方法代替DNA提取法虽然使敏感性降低5倍,但节省了DNA提取步骤,再结合显色技术使反应结果直观可见,使其临床应用更加便捷。

Five specific primers targeting 442-461 bp of the 16S rRNA gene of Riernerella anatipes- tifer were used to set up a LAMP reaction system. After DNA extraction and the addition of the DNA template,the LAMP reaction system was moved to a real-time turbidimeter （LA-200）, by which,the turbidity could be monitored during the reaction for 60 rain under 63℃. The sensitivities and specifieities of the LAMP with boiled bacteria solution versus extracted DNA and visualization versus agarose gel electrophoresis were also evaluated. Moreover, a total of 540 heart blood,brain,liver and heart samples collected from 135 ducks were assayed by LAMP and PCR after DNA extraction,then the results were compared with the results of Riernerella anatipestifer isolation, and the coincidence rates were calculated. The bacterium can be detected after reaction for 60 minutes at 63℃ ,with a sensitivity 5 times higher than PCR. As the result,the LAMP and PCR methods developed could detect as low as 2 and 10 CFU Riemerella anatipestifer respectively, when DNA was used as the template. And the detection limit was lowered down 5 times if the bacteria solution was treated by boiling for 10 min instead of DNA extraction. The sensitivity and spe-cificity of the three methods（visualization,turbidity and agarose gel eleetrophoresis） are the same.Totally 201 strains of Riemerella anatipestifer were isolated,and were confirmed by both LAMP and PCR. There are 271 and 254 samples were detected as positive by LAMP and PCR,respectively. So we could draw a conclusion that the sensitivity of LAMP was preferable to the PCR assay. Although its sensitivity was 5 times lower than DNA extraction, the boiling method and visualization save a lot time and make it very convenient to be used clinically.