艰难梭菌tcdA和tcdB基因绝对定量PCR方法的建立及应用  

Establishment and application of absolute quantitative PCR method for tcdA and tcdB genes of Clostridioides difficile

作　　者：古文鹏[1] 贾森泉 周永明[1] 尹建雯[1] 赵世文[1] 赵晓南[1] 吴媛[2] 伏晓庆[1] Gu Wenpeng;Jia Senquan;Zhou Yongming;Yin Jianwen;Zhao Shiwen;Zhao Xiaonan;Wu Yuan;and Fu Xiaoqing(Institute of Acute Infectious Disease Prevention and Control,Yunnan Centre for Disease Control and Prevention,Kunming 650500;Institute of the Communicable Disease Control and Prevention,Chinese Centre for Disease Control and Prevention,Beijing 102206)

机构地区：[1]云南省疾病预防控制中心急性传染病防制所,昆明650500 [2]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中国抗生素杂志》2025年第2期137-143,共7页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(No.82360398);云南省基础研究专项项目(No.202301AT070160);云南省技术创新人才培养对象项目(No.202405AD350003)。

摘　　要：目的建立基于SYBR染料法的能够精确定量艰难梭菌主要毒素tcdA和tcdB基因表达量的绝对定量PCR方法,并进行应用。方法以CD12038艰难梭菌参考菌株为研究对象,扩增tcdA和tcdB基因的部分片段,然后将二者克隆到pUC57质粒载体中进行测序鉴定。采用梯度稀释的重组质粒为标准品,SYBR荧光染料法进行qPCR扩增,建立标准曲线。然后在4种主要的产毒性艰难梭菌流行参考菌株中验证菌株培养后tcdA和tcdB基因的拷贝数。结果本研究建立了能够精确定量产毒性艰难梭菌tcdA和tcdB基因拷贝数的绝对定量PCR方法,其中tcdA基因的线性范围为(2.71×10^(2)~2.71×10^(9))拷贝/μL,灵敏性为(324.70±33.42)拷贝/μL,批内变异系数(CV)为0.60%~1.12%,批间CV为0.57%~0.92%。tcdB基因的线性范围为(2.59×10^(2)~2.59×10^(9))拷贝/μL,灵敏性为(539.05±133.28)拷贝/μL,批内CV为0.37%~1.61%,批间CV为0.84%~1.76%。2个基因的溶解曲线均呈现单一峰值,特异性良好。对4种产毒性艰难梭菌参考菌株培养24 h后的tcdA和tcdB基因拷贝数研究结果显示,R20291(ST1/RT027)菌株的tcdA和tcdB基因表达水平最高,而其余3个菌株的表达水平较为相似。结论本研究建立了基于产毒性艰难梭菌tcdA和tcdB基因的绝对定量PCR方法,该方法具有线性范围广、灵敏性高、特异性和可重复性良好等特点,为后续艰难梭菌主要毒素基因的精确定量研究提供了技术支持。Objective To establish an absolute quantitative PCR method that can accurately quantify the expression of toxic tcdA and tcdB genes in Clostridioides difficile based on the SYBR and carry out preliminary application.Methods The reference strain of C.difficile(CD12038)was used as the research object,and fragments of tcdA and tcdB genes were amplified,then both of them were cloned into the pUC57 plasmid vector for sequencing and identification.Gradient dilution of recombinant plasmid was used as the standard,and qPCR amplification was performed by the SYBR method to establish the standard curve.The copy numbers of the tcdA and tcdB genes were verified in four major toxic C.difficile epidemic reference strains after bacterial culture.Results The study established an absolute quantitative PCR method capable of accurately quantifying the copy number of the tcdA and tcdB genes,in which the linear range of the tcdA gene was 2.71×10^(2)~2.71×10^(9) copies/μL,the sensitivity was(324.70±33.42)copies/μL,and the coefficients of variation(CVs)within batch were 0.60%~1.12%,with inter-batch CVs of 0.57%~0.92%.The tcdB gene had a linear range of 2.59×10^(2)~2.59×10^(9) copies/μL,sensitivity of(539.05±133.28)copies/μL,intra-batch CV of 0.37%~1.61%,and inter-batch CV 0.84%~1.76%.The melt curves for both genes showed a single peak with good specificity.The results of the tcdA and tcdB gene copy numbers of the four prevalent C.difficile reference strains after 24 h of incubation showed that the R20291(ST1/RT027)strain had the highest expression level of both genes,while the other three strains showed no significant difference in both genes.Conclusions In this study,an absolute quantitative PCR method based on the tcdA and tcdB genes of C.difficile was established,which had the characteristics of a wide linear range,high sensitivity,good specificity and reproducibility.It provided technical support for the subsequent study of the precise quantification of major toxic C.difficile genes.

关 键 词：艰难梭菌 tcdA tcdB 绝对定量PCR 

分 类 号：R978.1[医药卫生—药品]