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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 establish a rapid and accurate PCR-HRM method for the identification of clinical mycobacterial species and provide new help for the early diagnosis and treatment of clinical mycobacterial infection . Methods 81 mycobacteria collected by traditional Roche culture method were collected. The 16S-23S rRNA internal transcribed spacer (ITS) was used as the target gene to synthesize the PCR-HRM method. The sequencing results as the gold standard, the results of PCR-HRM and traditional Roche culture method were compared. Results The correct identification rates of PCR-HRM and Roche were 100% and 95.1% respectively in 81 mycobacteria. There was no significant difference (P> 0.05) between PCR-HRM and Roche. The coincidence rates of the two methods were 100% for detecting mycobacterium tuberculosis (MTB), 100% for detecting non-mycobacterium tuberculosis (NTM) by PCR-HRM method, and NTM The coincidence rate was 92.9%. Conclusion Mycobacterium tuberculosis PCR-HRM identification method was initially established. This method can accurately identify common MTB and NTM, and may be a rapid identification method of clinical mycobacterium infection.