艰难梭菌毒素A和B双重环介导等温扩增检测方法的建立及验证  

Development and verification of a duplex loop-mediated isothermal amplification assay for simultaneous detection of toxin A and toxin B of Clostridium difficile

作　　者：席小燕 林玉兰 卢金莹 陈惠权 彭凌[2] XI Xiaoyan;LIN Yulan;LU Jinying;CHEN Huiquan;PENG Ling(Medical College,Shaoguan University,Shaoguan 512005,Guangdong Province,China)

机构地区：[1]韶关学院医学院,广东韶关512005 [2]韶关学院生物与农业学院,广东韶关512005

出　　处：《中国生物制品学杂志》2025年第1期67-72,79,共7页Chinese Journal of Biologicals

基　　金：2021年度韶关学院自然科学类项目(SY2021KJ08);韶关学院2023年度大学生创新创业训练计划项目(Sycxcy2023177);广东省自然科学基金(2017A030307041)。

摘　　要：目的建立快速检测艰难梭菌毒素A(Clostridium difficile toxin A,TcdA)和B(TcdB)的双重环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,为艰难梭菌(Clostridium difficile,CD)感染的快速检测和毒素分型提供技术支持。方法根据TcdA和TcdB基因的保守序列各设计1套LAMP引物,在同一体系内进行双重LAMP扩增,通过对反应体系和反应条件的优化,建立快速检测TcdA和TcdB的LAMP方法,对该方法进行特异性和灵敏度验证,并对LAMP扩增产物进行熔解曲线分析。用建立的方法对18份腹泻患者粪便样本进行检测。结果TcdA和TcdB均能进行较好扩增的双重LAMP反应条件为:Mg2+浓度6.8 mmol/L,dNTPs浓度1.2 mmol/L,扩增温度64℃。扩增结果可采用SYBR GreenⅠ染色完成初步检测,目标基因(菌)显示翠绿色荧光,6种其他常见肠道病原体(大肠埃希菌、屎肠球菌、粪肠球菌、产气荚膜梭菌、脆弱类杆菌、肉毒梭菌)无荧光;双重LAMP体系检测TcdA和TcdB的灵敏度分别为10~0和10~1个质粒/反应,通过分析熔解曲线的特征峰可判断CD菌株所含的确切目标毒素。18份腹泻患者粪便样本中有8份样本初步检测为阳性,经溶解曲线分析,均含TcdA和TcdB两种毒素。结论初步建立了TcdA和TcdB双重LAMP检测方法,该方法具有较好的灵敏度和特异性,可用于CD感染的快速检测,并可实现TcdA与TcdB的同时快速检测。Objective To develop a duplex loop-mediated isothermal amplification(LAMP)assay for rapid detection of Clostridium difficile toxin A(TcdA)and B(TcdB),so as to provide technical support for the rapid detection and toxin typing of Clostridium difficile(CD)infection.Methods According to the conserved sequences of TcdA and TcdB genes,two sets of LAMP primers were designed respectively,and duplex LAMP amplification was performed in the same system.By optimizing the reaction system and reaction conditions,a rapid LAMP detection method for TcdA and TcdB was developed.The specificity and sensitivity of this method were verified,and the melting curves of the LAMP amplified products were analyzed.The 18 stool samples from patients with diarrhea were detected by using the developed method.Results The duplex LAMP reaction conditions for good amplification of both TcdA and TcdB were as follows:Mg~(2+)concentration 6.8 mmol/L,dNTPs concentration 1.2 mmol/L,and amplification temperature 64℃.The amplification products were preliminarily detected by using SYBR Green I staining,and the target gene(bacteria)showed a bright green fluorescence,while the six other common intestinal pathogens(Escherichia coli,Enterococcus faecium,Enterococcus faecalis,Clostridium perfringens,Bacteroides fragilis,and Clostridium botulinum)exhibited no fluorescence.The sensitivity of the duplex LAMP system for detection of TcdA and TcdB was 10~0 and 10~1 plasmids/reaction respectively,and the exact target toxins contained in CD strains were determined by analyzing the characteristic peaks of the melting curves.Eight of 18 stool samples from patients with diarrhea were positive in preliminary detection,and the melting curve analysis showed that all of them contained TcdA and TcdB.Conclusion A duplex LAMP detection method for TcdA and TcdB has been developed with good sensitivity and specificity,which can be used for the rapid detection of CD infection and can realize the simultaneous rapid detection of TcdA and TcdB.

关 键 词：艰难梭菌毒素A 艰难梭菌毒素B 双重环介导等温扩增技术 毒素分型 

分 类 号：R378[医药卫生—病原生物学]