针对rcsA基因建立可视化LAMP方法快速检测肺炎克雷伯菌

Establishment of visualized LAMP assay targeting rcsA gene for rapid detection of Klebsiella pneumoniae

    摘要
  • 摘要:
    目的 建立一种可视化环介导等温扩增(LAMP)方法, 以快速检测肺炎克雷伯菌(KP), 并对该方法的检测灵敏度和特异性进行评价。
    方法 针对KP的rcsA基因设计4条特异性LAMP引物, 对反应条件(温度及时间)和反应体系中的名种参数进行优化, 并使用嵌合型荧光染料SYBR Green Ⅰ使反应可视化。通过染色可视化结果和琼脂糖凝胶电泳结果, 确定最佳反应条件和反应体系。利用优化后条件和体系, 以10倍梯度稀释DNA模板检测方法灵敏度, 同时对肺炎克雷伯菌和其他数十种临床常见细菌及真菌进行检测以评价其特异性。
    结果 优化后确定最佳反应温度为63 ℃、反应时间为35 min, 确定反应体系中缓冲液浓度为0.8×、镁离子浓度为8 mmol/L、dNTPs浓度为1.4 mmol/L、内外引物比为6∶1、Bst DNA聚合酶浓度为0.32 U/μl、甜菜碱浓度为0.75 mmol/L；灵敏度试验中, 该方法可检测的模板浓度可达1.5 ng/μl；特异性试验中, 仅肺炎克雷伯菌检测结果呈现阳性可视化结果及阳性电泳条带。
    结论 成功建立肺炎克雷伯菌可视化LAMP快速检测方法, 且特异性高、灵敏度高、结果易于观察、无需精密仪器, 临床检测初步应用成功后可适用于基层实验室检测。

     

    Abstract:
    OBJECTIVE To establish a visualized loop-mediated isothermal amplification (LAMP) for rapid detection of Klebsiella pneumoniae and evaluate the sensitivity and specificity of the detection method.
    METHODS Totally4 specific LAMP primers were designed by targeting to rcsA gene of the K. pneumoniae. The reaction conditions (temperature, time) and the parameters of the reaction system were optimized, the reaction was made visualized by using chimeric fluorescent dye SYBR GreenⅠ. The optimal reactions conditions and reaction systems were determined based on the results of visualization and agarose gel electrophoresis. The optimized conditions and systems were used to test the sensitivity by diluting DNA template on a 10-fold gradient, meanwhile, the specificity of the method was evaluated by detecting K. pneumoniae and other ten species of common bacteria and fungi.
    RESULTS The optimal reaction temperature was 63 ℃ after the optimization, with the reaction time 35 min; the concentration of buffer solution in the reaction system was determined as 0.8×, with the concentration of magnesium ion 8 mmol/L, the concentration of dNTPs 1.4 mmol/L, the ratio of internal to external primers 6: 1, the concentration of Bst DNA polymerase 0.32 U/μl, the concentration of betaine 0.75 mmol/L. For the test of sensitivity, the method could detect the template with the concentration of 1.5 ng/μl. For the test of specificity, only the detection of K. pneumoniae could display positive visualized result and positive electrophoretic band.
    CONCLUSIONS The visualized LAMP with high specificity and sensitivity for rapid detection of K. pneumoniae is successfully established, facilitating the observation of the detection result without the use of precise instruments. It is suitable for the detection in grass-root laboratories after successful preliminary application in clinical detection.

     

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