噬菌体生物扩增法、DNA序列分析及单链构象多态性分析检测结核分枝杆菌耐药性

目的　探讨和评价噬菌体生物扩增技术 (phageamplifiedbiologicallyassay,PhaB)用于检测结核分枝杆菌耐药性的应用价值。方法　本研究共选取了 91株利福平 (RFP)耐药株 (其中 82株为耐多药结核分枝杆菌 )、4 2株RFP敏感株、75株氧氟沙星 (OFLX)耐药株、4 0株OFLX敏感株 ,应用PhaB、DNA序列分析法和聚合酶链反应 单链构象多态性分析 (PCR SSCP) 3种检测方法 ,同时对同一菌株应用该 3种方法进行耐药性检测 ,以资分析与评价PhaB法的临床应用价值。结果　以绝对浓度法药敏试验结果为标准 ,检测RFP的药物敏感性 ,PhaB法的准确性为 93% ,敏感性为 92 % ,特异性为95 % ;DNA序列分析法的准确性为 93% ,敏感性为 90 % ,特异性为 10 0 % ;PCR SSCP法的准确性为90 % ,敏感性为 86 % ,特异性为 10 0 %。检测OFLX(OFLX)的药物敏感性 ,PhaB的准确性为 95 % ,敏感性为 95 % ,特异性为 95 % ;DNA序列分析法的准确性为 80 % ,敏感性为 71% ,特异性为 98% ;而PCR SSCP法的准确性为 75 % ,敏感性为 6 3% ,特异性为 98%。结论　PhaB法是一种操作简便、快速、敏感、与绝对浓度法符合率高的结核分枝杆菌耐药性检测方法 ,在 4 8～ 96h内可获得试验结果 ,具有良好的临床应用前景。

Analysis and evaluation of phage amplified biologically assay,DNA sequencing analysis and single-strand conformational polymorphism for the drug susceptibility testing of Mycobacterium tuberculosis

Objective To rapidly identify drug resistant Mycobacterium tuberculosis using phenotypic and genotypic methods and to evaluate the clinical significance of rapid phenotypic susceptibility test by phage amplified biologically assay(PhaB) Methods PhaB, DNA sequencing and polymerase chain reaction single strand conformational polymorphism(PCR SSCP)were performed on the 91 rifampicin(RFP) resistant strains(including 82 multi drug resistant Mycobacterium tuberculosis strains), 42 RFP susceptible strains, 75 ofloxacin(OFLX) resistant strains and 40 OFLX susceptible strains at the same time Results The results obtained using PhaB assay,DNA sequencing and PCR SSCP were compared with the absolute concentration method For RFP susceptibility,the accordance, sensitivity and specificity of PhaB were 93%,92% and 95% respectively;the accordance, sensitivity and specificity of DNA sequencing were 93%,90% and 100% respectively;those of PCR SSCP were 90%,86% and 100% respectively For OFLX susceptibility, the accordance, sensitivity and specificity of PhaB assay were 95%,95% and 95%;those of DNA sequencing were 80%,71% and 98% respectively;those of PCR SSCP were 75%,63% and 98% respectively Conclusions PhaB assay is a low cost, rapid, and sensitive method and shows high accordance with absolute concentration technology It can give drug susceptibility test results within 48 96 h, and is a promising technology in clinical laboratory