Tang Zhihui, Fu Lifa, Zhang Yanrong, Zhou Boyan, Feng Tianqin, Yang Wenjuan, Liang Ge, Yan Qianya, Zheng Canlin, Bie Mingjiang, Wang Baoning
（ 610041） West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China.
（ 610041） West China School of Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2024 Sep 20;55(5):1316-1321. doi: 10.12182/20240960402.
To establish a viable bacteria assay for () by assessing the gene expression, and to develop accordingly a rapid and novel testing method for clinical precision treatment.
Viable bacteria count was determined in bacterial cultures. The transcriptional expression level of (), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in , was measured by RT-PCR. The correlation between the number of colonies and 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.
The Ct values of for colony counts of 10, 10, 10, and 10 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 10-10 CFU/mL, the regression equation for gene expression and viable bacterial counts determined by RT-qPCR was =-0.3501+12.49, with the correlation coefficient being =0.9992 and the sensitivity being 10 CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live 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 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 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 (>0.05).
The method for assessing viable bacteria account by evaluating gene expression in was successfully established, significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.
通过评估（）基因表达建立一种可行的（）细菌检测方法，并据此开发一种用于临床精准治疗的快速新颖检测方法。
测定细菌培养物中的活菌数。通过RT-PCR测量（）中编码胆固醇-α-葡萄糖基转移酶（CGT）的保守基因（）的转录表达水平。分析菌落数与（）基因转录表达之间的相关性并构建回归模型。相应地检测新方法的线性范围、灵敏度和特异性。使用该方法评估克拉霉素的杀菌作用，以验证该方法在确定临床细菌耐药性方面的性能。
对于10、10、10和10 CFU/mL的菌落计数，（）的Ct值分别为29.67±0.14、23.37±0.36、17.65±0.37和11.38±0.39。在10 - 10 CFU/mL范围内，RT-qPCR测定的（）基因表达与活菌数的回归方程为=-0.3501 + 12.49，相关系数为=0.9992，灵敏度为10 CFU/mL，与其他13种细菌无交叉反应。用0、5、10、20和40 μg/mL克拉霉素处理12 h的活（）细菌的lg值分别为2.57±0.02、2.45±0.01、2.19±0.02、1.91±0.07和1.33±0.05。相应的（）基因Ct值分别为27.76±0.09、28.37±0.24、29.51±0.14、30.11±0.12和31.66±0.11。通过将（）基因表达应用于方程，发现估计的活菌数为2.73±0.03、2.52±0.08、2.11±0.05、1.89±0.02和1.33±0.04，统计分析无显著差异（>0.05）。
成功建立了通过评估（）中（）基因表达来评估活菌数的方法，显著缩短了测定活菌数所需的时间，并为临床活菌检测提供了一种新方法。
