空肠弯曲杆菌LAMP检测方法的建立及初步应用

目的 建立一种灵敏的环介导等温扩增方法（Loop-mediated Isothermal Amplification, LAMP）用于空肠弯曲杆菌(Campylobacter jejuni)的快速检测。方法：根据已公布的空肠弯曲杆菌旋转酶基因（gyrA）设计引物，建立并优化LAMP反应体系，对检测方法的特异性和灵敏度进行验证，并运用该方法对我国不同地方鸡种空肠弯曲杆菌的携带率进行调查。结果 该方法能扩增出典型的梯形条带，且扩增产物的酶切鉴定结果与理论值相符；对单增李斯特菌等8株实验菌株进行检测，仅空肠弯曲杆菌的LAMP结果为阳性；该方法检测空肠弯曲杆菌纯培养物的灵敏度为20cfu／mL，高于常规PCR检测方法10倍；不同地方鸡种空肠弯曲杆菌携带率介于6%～90%之间，差异显著。结论 本试验建立的空肠弯曲杆菌LAMP检测方法特异性强、灵敏度高，有望在临床样品检测中发挥作用。

Establishment and application of Loop-Mediated Isothermal Amplification method for Rapid Detection of Campylobacter jejuni

Objective To establish a method of Loop-mediated isothermal Amplification (LAMP) for detection of Campylobacter jejuni. Methods According to Campylobacter jejuni gyrA gene sequences published on GenBank, we designed specific loop-mediated isothermal amplification primers and developed a novel and highly specific loop-mediated isothermal amplification assay for the sensitive and rapid detection of Campylobacter jejuni. Their Campylobacter jejuni natural infection rates were tested in ten chicken breeds. Results The products of LAMP demonstrated the typical ladder patterns and digested fragments were found with the predicted sizes. Sensitivity of the LAMP asssay for direct detection of Campylobacter jejuni in pure cultures was 20cfu/mL. It was 10-fold more sensitive than that of the conventional PCR assay. The assay correctly identified Campylobacter jejuni, but did not detect 7 non-Campylobacter jejuni strains. The Campylobacter jejuni natural infection rate was 6%-90% in the ten chicken breeds. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of Campylobacter jejuni and will play a role in clinical detection.