产气荚膜梭菌TaqMan实时荧光定量PCR特异性引物设计和方法验证
Establishment and application of a TaqMan-based real-time fluorescence quantitative PCR detection method for Clostridium perfringens
马嘉琦 1孙波 1马红梅 1王东 1赵志军 2李勇 1曾瑾1
作者信息
- 1. 宁夏大学西部特色生物资源保护与利用教育部重点实验室,宁夏银川 750021;宁夏大学生命科学学院,宁夏银川 750021
- 2. 宁夏医科大学总医院宁夏临床病原微生物重点实验室,宁夏银川 750021;宁夏医科大学总院医学实验中心,宁夏银川 750021
- 折叠
摘要
目的 建立针对水样中产气荚膜梭菌检测的TaqMan实时荧光定量聚合酶链式反应(PCR)方法,并测试该方法在自来水样中的检测效果.方法 选择位于该菌拟核中高度保守的plc基因,设计特异性引物和TaqMan 探针,经优化后建立了针对该菌的TaqMan实时荧光定量PCR检测方法,结合滤膜法处理含有plc基因的标准菌株的模拟污染水样,并对所建立的方法进行测试.结果 所建立的产气荚膜梭菌TaqMan实时荧光定量PCR检测方法具有高度的特异性,13株食源性致病菌、3株艰难梭菌及1株腐败梭菌的Ct值大于40;该方法的最低检出限为1× 10copies/µL,具有较高的灵敏性;对模拟污染水样的最低检测限为1.0×102 CFU/mL.应用该方法对4份人工模拟污染阳性水样与90份自来水样进行检测发现,2份1.0×102CFU/mL的模拟污染水样可检出产气荚膜梭菌,2份1.0×10CFU/mL的模拟污染水样与90份自来水样均未检出产气荚膜梭菌.结论 所建立的产气荚膜梭菌TaqMan实时荧光定量PCR检测方法具有特异性好、灵敏性高的优点,对水体中产气荚膜梭菌的检测具有较高的应用价值.
Abstract
Objective A TaqMan-based real-time fluorescence quantitative polymerase chain reaction(PCR)method was established to detect Clostridium perfringens(C.perfringens)in tap water samples.Methods The highly conserved plc gene located in the pseudonucleus of the bacterium was amplified and specific primers and TaqMan probes were designed.After optimization,a TaqMan-based real-time fluorescence quantitative PCR detection method was established.Combined with the filter membrane method,simulated polluted water samples of standard strains containing plc genes were treated,and the established method was tested.Results The established TaqMan-based method for detecting C.perfringens showed high specificity.Ct＞40 were found in 13 foodborne pathogens,3 Clostridium difficile and 1 Clostridium putrefaciens strain.The detection limit of this method was 1×10 copies/µL,showing high sensitivity.The minimum detection limit for the simulated polluted water sample was 1.0×102 CFU/mL.This method was also used to detect 4 simulated water contamination samples and 90 imported water samples.The results showed that C.perfringens could be detected in 2 simulated 1.0×102 CFU/mL-contaminated water samples,while not in 2 simulated 1.0×10 CFU/mL-contaminated and imported water samples.Conclusion The TaqMan real-time fluorescence quantitative PCR method established in this study for the detection of C.perfringens has good specificity,high sensitivity,and practical value for detecting C.perfringens in water samples.
关键词
产气荚膜梭菌/实时荧光定量PCR/TaqMan探针法/水样检测/食源性致病菌Key words
Clostridium perfringens/real-time fluorescent quantitative PCR/TaqMan probe method/water sample detection/foodborne pathogens引用本文复制引用
基金项目
宁夏回族自治区自然科学基金重点项目(2022AAC02019)
宁夏回族自治区重点研发计划重点项目(2021BEF02028)
出版年
2024
NETL
NSTL
万方数据