| 于昌萍,于 彬,蔡为为,刘佰成,于 淼,王东亮.逆转录-聚合酶螺旋反应检测结核分枝杆菌的应用[J].,2020,(5):879-884 |
| 逆转录-聚合酶螺旋反应检测结核分枝杆菌的应用 |
| Application of Reverse Transcription-Polymerase Spiral Reaction for Detection of Mycobacterium Tuberculosis |
| 投稿时间：2019-08-28 修订日期：2019-09-23 |
| DOI：10.13241/j.cnki.pmb.2020.05.017 |
| 中文关键词: 结核分枝杆菌 聚合酶螺旋反应 临床样本 快速检测 |
| 英文关键词: Mycobacterium tuberculosis Polymerase spiral reaction Clinical samples Rapid detection |
| 基金项目:辽宁省自然科学基金项目（201510274-301）；辽宁省科学事业公益基金项目（2015001021） |
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| 中文摘要: |
| 摘要 目的：建立并应用逆转录-聚合酶螺旋反应（RT-PSR）快速检测结核分枝杆菌（MTB）。方法：针对结核分枝杆菌16S rRNA基因设计特异性引物，通过反应条件的优化初步建立结核分枝杆菌的RT-PSR扩增方法；随后，用2株结核分枝杆菌和11株其他致病菌进行RT-PSR、RT-LAMP和荧光定量PCR法的特异性与敏感性试验；利用RT-PSR法、罗氏培养法、RT-LAMP法和荧光定量PCR法对83名结核病患者的痰液标本进行诊断对比。结果：成功建立并优化了MTB的RT-PSR检测方法，与RT-LAMP法相比，两者特异性均为100%；RT-PSR法的检测灵敏性为1 CFU/mL，为荧光定量PCR法的10倍，且检测下限可达0.1 pg/μL；临床患者痰液样本检测结果表明，与罗氏培养法相比，RT-PSR法和RT-LAMP法的阳性率分别为98.80 %（P＜0.05）和96.39 %（P＜0.05），差异均具有统计学意义。结论：与传统检测法相比，RT-PSR法对于诊断临床样本中的MTB具有良好的特异性和敏感性，适合基层医疗单位防治MTB的推广和应用。 |
| 英文摘要: |
| ABSTRACT Objective: Establishment and application of reverse transcription-polymerase spiral reaction (RT-PSR) for rapid detection of Mycobacterium tuberculosis (MTB). Methods: The RT-PSR specific primers were designed by the 16S rRNA gene of M. tuberculosis and preliminary establishment of MTB amplification technology by optimizing the reaction conditions. Subsequently, the specificity and sensitivity of RT-PSR, RT-LAMP and real-time PCR method were tested with 2 strains of MTB and 11 other pathogenic bacteria. The diagnostic comparison of sputum samples from 83 tuberculosis patients by RT-PSR, Lowenstein-Jensen culture, RT-LAMP and real-time PCR method. Results: The RT-PSR method for MTB detection was successfully established and optimized. Compared with RT-LAMP method, the specificity of both methods was 100%. The sensitivity of RT-PSR was 1 CFU/mL, which was 10 times higher than that of real-time PCR and the detection limit was 0.1 pg/μL. The results of clinical samples showed that the positive rates of RT-PSR and RT-LAMP method were 98.80% (P<0.05) and 96.39% (P<0.05), and the difference was statistically significant when compared with Lowenstein-Jensen culture method. Conclusion: Compared with the traditional detection method, the RT-PSR method show good specificity and sensitivity for the diagnosis of MTB in clinical samples, which is suitable for the promotion and application of MTB prevention and treatment in primary medical units. |
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