高分辨溶解曲线技术鉴定常见致病分枝杆菌研究

Study of High Resolution Melting Identify Common Mycobacteria

目的建立快速、准确鉴定临床常见分枝杆菌菌种的聚合酶链高分辨溶解曲线(polymerase chain reaction high resolution melting,PCR-HRM)分析方法,为临床分枝杆菌感染的早期诊断和治疗提供新帮助。方法收集传统罗氏培养法的81株分枝杆菌。以16S-23S rRNA内转录间隔区(internal transcribed spacer,ITS)为靶基因合成引物,建立PCR-HRM方法。以测序结果为金标准,将PCR-HRM法和传统罗氏培养法鉴定的结果进行对比。结果 81株分枝杆菌中,PCR-HRM法和罗氏培养法的正确鉴定率分别为100%、95.1%,PCR-HRM法和罗氏培养法的正确鉴定率无统计学差异(P>0.05)。两种方法检测结核分枝杆菌(mycobacterium tuberculosis,MTB)的符合率均为100%,PCR-HRM方法检测非结核分枝杆菌(nontuberculosis mycobacteria,NTM)的符合率为100%,罗氏培养法检测NTM的符合率为92.9%。结论初步建立了分枝杆菌PCR-HRM鉴定方法,该方法可准确鉴定常见的MTB和NTM,可能成为临床分枝杆菌感染的快速鉴定方法。

Objective To develop a rapid and accurately species identifying method for mycobacteria by using polymerase chain reaction high resolution melting（ PCR-HRM） analysis and to offer the new help for early diagnosis and treatment of clinical mycobacteria infection. Methods A selection of 81 clinical mycobacteria isolates were tested in this study. Primers were synthesised according to 16S-23 S rRNA internal transcribed spacer（ ITS） and mycobacteria isolates were identified by PCR-HRM method. The sequencing results were considered as gold standard. To compare the results of PCR-HRM method and traditional Lowenstein-Jensen culture method. Results This study established PCR-HRM method for mycobacteria by means of optimizing the reaction system and reaction program. Among the 81 isolates,PCR-HRM method and Lowenstein-Jensen culture method was found 100% and 95. 1% correct identification,respectively. There was no statistical difference between two methods（ P 0. 05）. The consistency rate of two methods for testing mycobacteriam tuberculosis（ MTB） were all 100%. The consistency rate of PCR-HRM method and Lowenstein-Jensen culture method for testing nontuberculosis mycobacteria（ NTM） were100% and 92. 9%. Conclusion PCR-HRM method for MTB has established which can accurately identify common MTB and NTM. PCR-HRM analysis may be the rapid identifying method for MTB.