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10^(2)、10^(4)、10^(6)、10^(8) 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10^(1)~10^(8) 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10^(1) CFU/mL,此方法与13种其他菌无交叉反应。用0、5、10、20、40μg/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。
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 RTPCR.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 10^(2),10^(4),10^(6),and 10^(8) 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^(1)-10^(8) 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 10^(1) 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 40μg/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.
作者
唐智慧
符立发
张焱荣
周博彦
冯天勤
杨文娟
梁鸽
严茜雅
郑璨璘
别明江
王保宁
TANG Zhihui;FU Lifa;ZHANG Yanrong;ZHOU Boyan;FENG Tianqin;YANG Wenjuan;LIANG Ge;YAN Qianya;ZHENG Canlin;BIE Mingjiang;WANG Baoning(West China School of Basic Medical Sciences and Forensic Medicine,Sichuan University,Chengdu 610041,China;West China School of Medicine,Sichuan University,Chengdu 610041,China;West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu 610041,China;Editorial Office of Journal of Sichuan University(Medical Sciences),Chengdu 610041,China)
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2024年第5期1316-1321,共6页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金项目(No.82260402)
西藏自治区科技厅基金项目(No.XZ202201ZY0009N)
四川省科技厅基金项目(No.2023YFQ0051、No.2024YFFK0224)资助。
关键词
幽门螺杆菌
cgt基因
基因表达量
活菌测定
Helicobacter pylori
cgt gene
Gene expression level
Viable bacteria assay
