普通TaqMan探针及raqMan MGB探针real-time PCR检测无形体msp-2基因方法的建立与比较

摘要

目的 建立敏感、特异、定量real-time PCR方法,用于无形体病早期诊断及立克次体病鉴别诊断.方法 通过GenBank Blast比较分析,选择世界各地39株无形体,以msp-2特异基因保守区设计普通TaqMan探针及TaqMan MGB探针并比较分析.结果 普通TaqMan探针及TaqMan MGB探针2种方法均具有良好的特异性,除无形体外,遗传亲缘关系较近的其他立克次体目成员(17种)均扩增阴性.临床上其他8种常见致病菌呈阴性扩增.灵敏性评估结果发现普通TaqMan探针及TaqMan MGB探针最低检出浓度分别为102 copies/μl及10 copies/μl.两种探针方法均具有较好的重复性.结论 本研究建立了敏感、特异的无形体早期诊断及立克次体鉴别诊断的荧光定量PCR技术,TaqMan MGB探针灵敏性高于普通TaqMan探针.%Objective In order to develop a highly specific and sensitive real-time PCR assay to detect and indentify Anaplasma, the agent of Anaplasmoses. Methods A specific msp-2 outer membrane protein gene of A.phagocytophilum was selected for designing general TaqMan probe and TaqMan MGB probe based on the conserved sequences of msp-2 genes of 39 isolates all over the world. The evaluation of assay included the specificity, lowest detection load (LOD) and stability. Results Similar specificity and reproducibility for the two real-time PCR mcthods with general TaqMan probe and TaqMan MGB probe were demonstrated. Besides A. phagocytophilum, 17 members of Rickettsia and other 8 common pathogens in clinical hospitals were not amplified. The LOD by PCR with TaqMan MGB probe was lO times than that by PCR with general TaqMan. Conclusion Two highly specific and sensitive real-time PCR assays were developed to test A. phagocyophilum infection during the early phase of illness, but the assay with TaqMan MGB probe is more sensitive than that with TaqMan probe.