空肠弯曲菌LAMP快速检测方法的建立

Establishment of Loop-mediated Isothermal Amplification (LAMP) Method for Campylobacter jejuni Detection

摘要:

本研究使用恒温环介导技术, 以空肠弯曲菌的促旋酶基因A(gyrA)设计引物, 建立空肠弯曲菌的LAMP快速检测方法。不同来源的4株空肠弯曲菌LAMP检测均显示阳性, 其他14种细菌LAMP检测显示阴性。实验结果表明, 设计的引物具有良好的特异性。本研究进行了LAMP检测方法与细菌平板计数法和PCR法的灵敏度比较, 结果LAMP检测与PCR法有相近的灵敏度, 比细菌平板计数法灵敏度高3个数量级。我们还研究发现提取核酸前加入DNase可以有效地减少死菌DNA对LAMP结果的影响。使用LAMP方法对鸡法氏囊的检测表明, 结合核酸提取步骤中的DNase处理步骤, 可以准确的检测出鸡法氏囊中的空肠弯曲菌。

关键词:恒温环介导, 空肠弯曲菌, 检测

Abstract:

A rapid LAMP detection method with primers designed on genus-specific region identified in the gyrA gene was established in this assay. All four Campylobacter jejuni from different sources were detected positive and fourteen non Campylobacter bacteria were negative, which shows excellent specificity of the primers. Compared with plate count and PCR method, the LAMP method and the PCR method had equal sensitivity, which were three orders of magnitude higher than plate count. In this assay, we also found out that the treatment of DNase could reduce the dead bacteria DNA effectively. The LAMP detection on chicken indicated relatively good result on detection of Campylobacter jejuni combining with treatment of DNase.

Key words:Loop-mediated isothermal amplification, Campylobacter jejuni, Detection