高通量测定技术检测结核分枝杆菌耐利福平的研究

目的 基于焦磷酸测序(pyrosequencing)技术，建立耐利福平结核分枝杆菌快速检测方法，并探讨其临床应用价值。方法 设计一对聚合酶链反应(PCR)引物，其中一引物经生物素化修饰，PCR扩增含耐药决定区一297bp长的rpoB基因片段，借用链亲和素包被磁珠纯化单链PCR产物。设计2个正向测序引物，运用pyrosequencing技术对耐药决定区进行序列测定。通过对一系列10倍稀释的结核分枝杆菌H37Rv标准株DNA进行分析，评价所建立方法的检测敏感性。通过对已知耐药表型的结核分枝杆菌临床分离株进行分析，评价所建立方法的检测特异性。结果：PCR扩增后，可在2h内得到耐药决定区序列。所建立方法的检测灵敏度为50fg DNA／反应。在所分析的利福平敏感株中均未检测到耐药决定区突变，而在耐利福平菌株中均检测到耐药决定区突变。结论 所建立的方法具有自动化程度高和结果准确等特点，适用于对耐利福平结核分枝杆菌进行快速高通量检测。

Rapid detection of rifampin resistance of Mycobacterium tuberculosis using high-throughput pyrosequencing technique

Objective To develop a pyrosequencing approach to rapid detection of rifampin resistance in Mycobacterium tuberculosis based on characterization of all possible mutations in the 81 bp core region. Methods Two pyrosequencing sequencing primers and 1 pair of PCR primers were chosen for pyrosequencing analysis. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from Mycobacterium tuberculosis H 37Rv strains. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical Mycobacterium tuberculosis isolates with known antibiotic phenotypes. Results Rifampin resistance could be determined within 2 hours after PCR amplification and single-stranded template preparation by using only two pyrosequencing reactions. About 50 fg DNA/reaction was required in order to get sufficient PCR product for producing a long, clear and accurate pyrosequencing pattern. A total of 41 Mycobacterium tuberculosis isolates were tested and the results were concordant with those based on drug susceptibility testing and conventional DNA sequencing. Conclusion The pyrosequencing technique is simple to perform and can be used as a rapid, high-throughput and efficient method for detecting rifampin resistance in Mycobacterium tuberculosis.