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幽门螺杆菌cgt基因表达量测定活菌的方法建立及应用研究

Viable Bacteria Assay of Helicobacter pylori by RT-qPCR Measurement of cgt Gene Expression Levels: Establishment and Application of a New Method

    摘要
  • 摘要:
    目的 本研究旨在建立一种通过幽门螺杆菌（Helicobacter pylori, H. pylori）cgt基因表达量来检测活菌的方法，以便为临床精准治疗开发快速、便捷的检测手段。
    方法 通过培养法对活菌计数，并利用RT-qPCR方法测定相应H. pylori胆固醇-α-葡萄糖基转移酶（cholesterol-α-glucosyltransferase, CGT）保守编码基因cgt （hp0421）的mRNA表达水平，分析菌落数与cgt基因表达量之间的相关性，并构建回归方程；探索该方法可测定的线性范围、灵敏度和特异性。将此方法用于测定克拉霉素杀菌作用，验证其在细菌耐药检测中有效性。
    结果 H. pylori菌落数为102、104、106、108 CFU/mL的cgt的Ct值分别是29.67±0.14、23.37±0.36、17.65±0.37、11.38±0.39，在H. pylori菌落数为101～108 CFU/mL范围内，RT-qPCR法测定的cgt基因表达量和活菌数对应的回归方程： y=−0.3501x+12.49，决定系数R2=0.9992、灵敏度为101 CFU/mL，此方法与13种其他菌无交叉反应。用0、5、10、20、40 μg/mL质量浓度的克拉霉素作用12 h后培养法测定H. pylori的活菌lg值分别为2.57±0.02、2.45±0.01、2.19±0.02、1.91±0.07、1.33±0.05，对应cgt基因的Ct值分别为27.76±0.09、28.37±0.24、29.51±0.14、30.11±0.12、31.66±0.11，将cgt基因表达量代入上述方程推算活菌数为2.73±0.03、2.52±0.08、2.11±0.05、1.89±0.02、1.33±0.04，经分析差异无统计学意义（P>0.05）。
    结论 本研究成功建立了通过测定H. pylori的cgt基因表达量反映活菌的方法，有效缩短了检测周期，为临床活菌检测提供了新方法。

     

    Abstract:
    Objective To establish a viable bacteria assay for Helicobacter pylori (H. pylori) by assessing the cgt gene expression, and to develop accordingly a rapid and novel testing method for clinical precision treatment.
    Methods Viable bacteria count was determined in bacterial cultures. The transcriptional expression level of cgt (hp0421), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in H. pylori, was measured by RT-PCR. The correlation between the number of colonies and cgt gene transcription expression was analyzed and the regression model was constructed. The linear range, sensitivity, and specificity of the new method were examined accordingly. The bactericidal action of clarithromycin was assessed using this method to verify the performance of the method in determining clinical bacterial drug resistance.
    Results The Ct values of cgt for H. pylori colony counts of 102, 104, 106, and 108 CFU/mL were 29.67±0.14, 23.37±0.36, 17.65±0.37, and 11.38±0.39, respectively. In the range of 101-108 CFU/mL, the regression equation for cgt gene expression and viable bacterial counts determined by RT-qPCR was y=−0.3501x+12.49, with the correlation coefficient being R2=0.9992 and the sensitivity being 101 CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live H. pylori bacteria treated with clarithromycin at 0, 5, 10, 20, and 40 μg/mL for 12 h were 2.57±0.02, 2.45±0.01, 2.19±0.02, 1.91±0.07, and 1.33±0.05, respectively. The corresponding cgt gene Ct values were 27.76±0.09, 28.37±0.24, 29.51±0.14, 30.11±0.12, and 31.66±0.11. By applying the cgt gene expression in the equation, the estimated counts of viable bacteria were found to be 2.73±0.03, 2.52±0.08, 2.11±0.05, 1.89±0.02, and 1.33±0.04, showing no significant difference in statistical analysis (P>0.05).
    Conclusion The method for assessing viable bacteria account by evaluating cgt gene expression in H. pylori was successfully established, significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.

     

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