应用变性高效液相色谱法对蜱传立克次体的鉴定和分型

Identification and classification on tick-borne Rickettsia by PCR-DHPLC

目的建立PCR-DHPLC检测法,对5种蜱传人类致病性立克次体进行准确、快速的分型检测。方法针对蜱传5种致病性立克次体的Osp A为靶基因进行PCR扩增,本研究所用目的片段为合成片段,连接于18-T载体,选用1对特异性通用引物扩增此片段。扩增产物长度分别为513 bp、533 bp、533 bp、533 bp、533 bp。应用42株菌株进行特异性实验,建立PCR-DHPLC反应体系,并将此方法应用到实际样本检测。结果应用PCR-DHPLC技术可很好的区分5种致病菌,在核酸水平上达0.01 pg/μl。实际样本的峰型与标准峰型有较好对应性。结论 PCR-DHPLC法在实际样本检测中具有灵敏性和特异性。

Objective In order to establish the detection method of PCR-DHPLC, The Rickettsia from 5 kinds of tick were detected accurately and quickly applying for PCR-DHPLC technology. Methods Rickettsia Osp A from 5kinds of tick as the target gene was amplified by PCR. The synthesized fragment was connected with the 18-T vector. 1 pair of specific primer was used for amplifying the fragment. The lengths of product were 513 bp, 533 bp,533 bp, 533 bp and 533 bp. In the application of 42 strains, the specific experiments were carried out to establish the PCR-DHPLC reaction system, and the method was applied to the field collected sample detection. Results The application of PCR-DHPLC can be well separated 5 kinds of pathogenic Rickettsia, at the nucleic acid level of 0.01pg/ul. The peak and the standard of the sample have good correspondence. Conclusion The PCR-DHPLC method is established which is sensitiv and specific.