啮齿动物螺杆菌多重PCR检测方法的建立

Establishment of a Multiplex PCR Procedure for Detection of Rodent Helicobacters

目的建立一种可同时检测肝、胆汁、啮齿类三种螺杆菌的多重PCR方法。方法根据已公布的肝、胆汁、啮齿类三种螺杆菌16SrRNA基因序列设计三对特异性引物进行多重PCR并对反应条件进行优化。结果三对引物能分别扩增出特异性的417 bp、364 bp、324 bp目的条带。最佳退火温度为52℃,镁离子浓度为2.0mmol/L,dNTP浓度为200μmol/L,引物浓度为0.625μmol/L。在此条件下多重PCR同时检测的肝、胆汁、啮齿类三种螺杆菌敏感度均为10 fg。结论本实验建立的多重PCR是一种敏感、特异、高效的方法,为同时检测啮齿动物中肝、胆汁、啮齿类三种螺杆菌奠定了良好的基础。

Objective To establish a multiplex PCR procedure for simultaneous detection of H.hepaticus,H.bilis,and H.rodentium,and to preliminarily verify its application value.Methods Three pairs of specific primers for the multiplex PCR procedure were designed according to published H.hepaticus,H.bilis,and H.rodentium 16S rRNA gene sequences,and the reaction conditions of the multiplex PCR were optimized.Results The results showed that all the three pairs of primers could be used to specifically amplify their target bands of 417 bp,364 bp and 324 bp.The optimal annealing temperature was 52℃,the Mg2＋ concentration was 2.0 mM,the dNTP concentration was 200 μΜ,and the concentration of primers was 0.625 μM.The sensitivity of the multiplex PCR for H.hepaticus,H.bilis,H.rodentium was 10 fg.Couclusions A sensitive,specific,efficient multiplex PCR procedure has been established in this study,providing a good foundation for the detection of H.hepaticus,H.bilis,and H.rodentium.