摘要
目的建立一种灵敏、可靠、快速对水体中致病菌富集和核酸提取的前处理方法,用于水体致病菌荧光定量PCR检测。方法通过加标准菌株脓肿分枝杆菌、原菌液梯度稀释,采用离心柱法和磁珠法核酸提取试剂盒结合不同滤膜洗脱方式进行核酸提取;采用不同材质、尺寸、数量的击打物质及均质仪循环次数等对核酸提取条件进行优化;所得核酸进行荧光定量PCR检测,比较各方法的提取效率;采用最优提取条件,对加标水样进行荧光定量PCR检测,考察全流程回收率和检出限。结果采用0.20μm滤膜过滤+7颗大锆珠均质仪循环6次+TIANMicrobe Magnetic Envir-DNA Kit方法回收率最高(81.03%±19.23%),检出限达到6.20 CFU/100 mL。结论均质仪+TIANMicrobe Magnetic Envir-DNA Kit法更适用于水体中致病菌的富集和核酸提取。
Objective To establish a sensitive,reliable and rapid pre-treatment method for enrichment and nucleic acid extraction of pathogenic bacteria in water for fluorescent quantitative PCR(qPCR)detection of pathogenic bacteria in water.Methods The tester strain used in the experiment was Mycobacterium abscessus(American Type Culture Collection[ATCC]19977)at concentration of 3.10×10^(8)colony form unit(CFU)/mL.The nucleic acid of the tester strain was extracted using centrifugal column and magnetic bead extraction kits combined with elutions of different membranes.The nucleic acid extraction conditions were optimized by using beads of different materials,sizes and numbers and the number of homogenization cycles.The extracted nucleic acid was subjected to qPCR assay to compare the extraction efficiency of each method.qPCR was performed on spiked water samples using optimal extraction conditions to examine the whole process recovery and detection limits.Results The highest recovery of 81.03%±19.23%and detection limit of 6.20 CFU/100 mL were achieved using the procedures including 0.20μm membrane filtration,six homogenization cycles with seven large zirconium beads,and TIANMicrobe Magnetic Envir-DNA Kit based extraction.Conclusion Using homogenizer and TIANMicrobe Magnetic Envir-DNA Kit is more efficient for the enrichment and nucleic acid extraction of pathogenic bacteria in water for qPCR detection.
作者
吴妍
张晓
张良
邢方潇
张岚
WU Yan;ZHANG Xiao;ZHANG Liang;XING Fangxiao;ZHANG Lan(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases/Department of Water Quality and Population Health Surveillance,National Institute of Environmental Health,Chinese Center for Disease Control and Prevention,Beijing 100050,China)
出处
《中国公共卫生》
CAS
CSCD
北大核心
2023年第11期1496-1500,共5页
Chinese Journal of Public Health
基金
国家重点研发计划(2021YFC3200805–5)
国家重点研发计划(2022YFC3204702)
关键词
致病菌
水体
核酸提取
荧光定量PCR
pathogenic bacteria
water environment
nucleic acid extraction
fluorescent quantitative PCR
