论文部分内容阅读
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。
A loop-mediated isothermal amplification (LAMP) reaction system was established by using 5 specific primers corresponding to 442 ~ 461 bp of 16S rRNA gene of Ralstonia eutropha. Reaction 60min, by monitoring the reaction solution turbidity to determine the results. On this basis, the method of boiling 10min instead of kit DNA extraction method, and the colorimetric method instead of turbidity detection and agarose gel electrophoresis method to determine the results through the sensitivity, specificity, pathogen growth and decline of the law to verify the accuracy of the method Sex. In addition, a total of 540 samples of blood, brain, liver and heart collected from 135 diseased ducks were collected and DNA was extracted by LAMP and PCR. Meanwhile, bacterial isolation and identification were performed. The results of various methods were compared with each other rate. The results showed that 2 and 10 CFU of Ralstonia solani were detected by LAMP and PCR respectively after the DNA was extracted from the bacteria. Bacteria boiling instead of kit DNA extraction method, the sensitivity of LAMP reduced by 5 times. The sensitivities and specificities of the results are judged by colorimetry, turbidity detection and agarose gel electrophoresis. A total of 201 strains of Ralstonia eutropha were obtained by bacterial isolation method, and all of them were positive by LAMP and PCR. The total number of positive samples detected by LAMP and PCR was 271 and 254, respectively. The positive rate of LAMP was higher than that of PCR . The established method of LAMP detection of Ralstonia emersonii has high sensitivity. However, using DNA extraction method instead of DNA extraction method to reduce the sensitivity by 5 times, but save DNA extraction step, combined with color technology to make the reaction result Intuitively visible, to make it more convenient clinical application.