Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil.
Mem Inst Oswaldo Cruz. 2013 Feb;108(1):106-9. doi: 10.1590/s0074-02762013000100017.
Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.
采用实时荧光定量聚合酶链反应-高分辨率熔解（qPCR-HRM）分析方法筛选结核分枝杆菌耐药相关突变。我们使用基于质粒的对照物，通过 qPCR-HRM 检测 rpoB 基因中利福平耐药决定区（RRDR）的 C526T 和 C531T 突变。从结核分枝杆菌 H37Rv 和携带 rpoB 中 C531T 或 C526T 突变的菌株中克隆 RRDR 区的一个片段到 pGEM-T 载体中，并将这些载体用作 54 株结核分枝杆菌中 qPCR-HRM 分析的对照。结果通过 DNA 测序得到证实，表明重组质粒可替代基因组 DNA 作为 qPCR-HRM 检测的对照。质粒可在生物安全 3 级设施外处理，降低污染风险和检测成本。质粒稳定性高，通常在大肠杆菌中维持，并可大量提取。
