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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 To establish a PCR-DHPLC detection method for accurate and rapid genotyping of five tick-borne rickettsial pathogens. Methods The target gene of Osp A was amplified by PCR from five pathogenic rickettsiae of ticks. The target fragment used in this study was a synthetic fragment and ligated to 18-T vector. A pair of specific universal primers was used to amplify this Fragment. The length of amplified products were 513 bp, 533 bp, 533 bp, 533 bp and 533 bp, respectively. 42 strains of bacteria were used to carry out specific experiments to establish PCR-DHPLC reaction system, and this method was applied to the actual sample test. Results Using PCR-DHPLC technique, five pathogenic bacteria were well differentiated and reached 0.01 pg / μl at the nucleic acid level. The actual sample peak shape and standard peak type has a good correspondence. Conclusion The PCR-DHPLC method is sensitive and specific in the detection of real samples.