结核分枝杆菌链霉素耐药性的噬菌体检测技术研究

Study on rapid detective technique for streptomycin resistance in Mycobacterium tuberculosis by phage amplified biologically assay

目的建立结核分枝杆菌(结核菌)链霉素耐药性的噬菌体快速检测技术,并探讨其临床应用价值。方法应用分枝杆菌噬菌体感染结核菌建立链霉素耐药性的噬菌体检测技术,并对最佳测定条件进行探讨;将所建立的方法用于38株世界卫生组织药敏质控株和372株临床分离株链霉素耐药性测定,并与绝对浓度法和Bactec960药敏结果比较,对不符合菌株进行最低抑菌浓度(MIC)和rpsL耐药基因检测。结果链霉素终浓度5μg/ml作用24h、噬菌体108噬菌体形成单位/ml感染60min、杀毒剂5%室温作用5min为选择的检测条件。噬菌体法检测38株药敏质控株结果完全符合。372株临床分离株链霉素耐药性检测结果表明,如以绝对浓度法药敏结果为判断标准,则本法敏感性为97.4%、特异性为91.0%、阳性预测值为81.7%、阴性预测值为98.9%、准确性为92.9%;如以Bactec960药敏结果为判断标准,则本法敏感性为97.2%、特异性为97.1%、阳性预测值为94.6%、阴性预测值为98.5%、准确性为97.1%。有22株噬菌体法药敏结果与常规药敏试验结果不符,其中的11株噬菌体法检测结果与MIC和rpsL耐药基因检测结果相同。结论噬菌体法检测链霉素耐药性快速、简便,具有很高的敏感性和特异性,可作为结核菌链霉素耐药性的快速检测方法。

Objective To set up a rapid detective technique for streptomycin resistance in Mycobacterium tuberculosis （MTB） by phage amplified biologically （PhaB） assay and evaluates the value for application in clinical isolates. Methods The PhaB assay for detection of streptomycin resistance was established by infection of MTB with mycobacteriophage ; The test condition was discussed. Streptomycin resistance was detected in 38 quality control strains and 372 clinical isolates of MTB . The results was compared with that of the absolute concentration and BACTEC 960 methods. The minimum inhibitory concentration （MIC） and rpsL gene mutation was tested by liquid medium and PCR-SSCP methods for all discrepant isolates. Results The PhaB assay was detected with the following test conditions ： Streptomycin 5 μg/ml, 37℃ for 24 h; mycobacteriophage 10^8 PFU/ml, 37℃ for 60min; virucidal concentration 5% for 5min at room temperature. The result for 38 quality control strains tested by PhaB assay was complete correlation with that of conventional method. If the results of absolute concentration method was taken as judgment standard the sensitivity, specificity, positive and negative predictive value as well as accuracy were 97.4%, 91.0%, 81.7%, 98.9% and 92. 9% respectively detected by PhaB assay. The sensitivity,specificity, positive and negative predictive value as well as accuracy was 97.2%, 97.1%, 94. 6%,98. 5% and 97. 1% respectively tested by PhaB assay compared with that of the BACTEC 960. A total of 22 strains were discrepant in comparison with the PhaB assay and conventional methods. The results of PhaB assay in 11 strains were same as MIC and gene mutation test among 22 discrepant strains. Conclusion Because its rapidity, simplicity, sensitivity and specificity, the PhaB assay can be used for rapid detection of streptomycin resistance in Mycobacterium tuberculosis.