基于hsp65基因的PCR-RFLP用于快速鉴定分枝杆菌菌种的初步研究

目的 基于hsp65基因的PCR-限制性酶切片段长度多态性(PCR-RFLP)用于快速鉴定分枝杆菌菌种的方法的建立和评价.方法 选择分枝杆菌hsp65基因作为目的 基因,对分枝杆菌标准株进行PCR扩增,扩增产物经HaeⅢ和Bstp Ⅰ两种限制性内切酶酶切,酶切产物采用4%MetaPhor琼脂糖凝胶电泳,根据电泳条带位置,计算条带分子量,确定不同菌种的特异指纹图谱.结果 共对40种(株)分枝杆菌进行分析,6株结核分枝杆菌复合体菌株呈现两种指纹图谱,34株非结核分枝杆菌标准株均具有惟一的指纹图谱.结论 基于hsp65基因的PCR-RFLP可用于分枝杆菌菌种的快速鉴定,且方法简便,易于标准化.

Preliminary study on rapid identification the species of Mycobacteria by PCR-restriction fragment length polymorphism based upon hsp65 gene

Objective To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level. Methods hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae Ⅲ and Bstp Ⅰ, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed. Results A total of 40 Mycobacterium species (strains) was analyzed, in which six reference strains of Mycobacterium tuberculosis Complex had two different electrophoresis patterns, and thirty-four reference species of non-tuberculosis Mycobacteria had unique pattern. Conclusion PCR-RFLP Based upon hsp65 gene can be used for identification of Mycobacterium species, and the method is more rapid and simple and easy-to-use for mycobacterial species identification.