多重微滴数字PCR同时定量检测三种食源性致病菌DNA拷贝数  

Simultaneous Quantification Method of DNA Copy Number for Three Food-borne Pathogens by Multiplex Droplet Digital PCR

作　　者：张明明 肖剑 林秀敏 尹玮璐 梁美丹 周露 孙雪奇 ZHANG Ming-Ming;XIAO Jian;LIN Xiu-Ming;YIN Wei-Lu;LIANG Mei-Dan;ZHOU Lu;SUN Xue-Qi(Guangzhou Institute for Food Inspection,Guangzhou 511406,China;Guangdong Institute of Food Inspection,Guangzhou 510407,China)

机构地区：[1]广州市食品检验所,广州511406 [2]广东省食品检验所,广州510407

出　　处：《农业生物技术学报》2022年第3期606-618,共13页Journal of Agricultural Biotechnology

基　　金：广州市科技计划项目(201904010253);广州市市场监督管理局科技项目(2020kj52);国家市场监督管理总局科技计划项目(2019NK090)。

摘　　要：食源微生物是影响食品质量与安全的重要因素,微滴数字PCR(droplet digital PCR,ddPCR)能够对核酸进行绝对定量检测,在食源性致病菌检测中具有越来越重要的地位。为实现食源性致病菌基因组拷贝数的快速绝对定量检测,本研究从已报道的伤寒沙门氏菌(Salmonella typhi)、金黄色葡萄球菌(Staphylococcus aureus)及单核细胞增生李斯特氏菌(Listeria monocytogenes)检测引物和探针中,经TaqMan探针法qPCR特异性验证,筛选获得以ttrA/ttrC、FMN-binding glutamate synthase、invasion associated endopeptidase基因为靶标的引物和探针,并将伤寒沙门氏菌和金黄色葡萄球菌检测探针连接6-羧基荧光素(6-carboxy-fluorescein,FAM)基团、单核细胞增生李斯特氏菌检测探针连接六氯-6-甲基荧光素(hexachloro fluorescein,HEX)基团,分别构建3种目标菌株的单重ddPCR检测体系。在此基础上,通过引物和探针浓度和退火温度的优化构建可在同一反应体系中同时、快速、绝对定量检测伤寒沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌基因组拷贝数的多重ddPCR检测体系。多重ddPCR检测体系可扩增获得8(23)个微滴簇区域,对伤寒沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏菌定量检测的线性范围分别为235~0.23、284~0.18、380~0.42 copies/µL;在定量检测范围内的线性相关系数R^(2)均大于0.999,且重复性较好,3种致病菌的多重ddPCR检测体系与单种菌的单重ddPCR定量线性范围几乎一致。本研究构建的多重ddPCR检测体系特异性强、稳定性好、定量范围广,可为食源性致病菌的无增菌绝对定量检测提供技术支持。Food-borne microorganisms are important factors affecting food quality and safety.Droplet digital PCR(ddPCR),which can be used for the absolute quantitative detection of nucleic acids,plays an increasingly important role in the detection of food-borne pathogens.In order to quantify the genome copy numbers of food-borne pathogens,a strategy for simultaneous detection of the DNA copy numbers of Salmonella typhi,Staphylococcus aureus and Listeria monocytogenes by multiplex droplet digital PCR(ddPCR)was developed in this study.Primers and probes targeting ttrA/ttrC、FMN-binding glutamate synthase、invasion associated endopeptidase gene were selected to evaluate the feasibility and applicability of the strategy by specific verification using TaqMan probe qPCR.The probes for Salmonella typhi and Staphylococcus aureus were labeled with 6-carboxy-fluorescein(FAM)fluorophore,and the probe for L.monocytogenes was labeled with hexachloro fluorescein(HEX)fluorophore.The detection systems of simplex ddPCR for 3 target strains were constructed.On this basis,through the optimization of primer and probe concentration and annealing temperature,a multiplex ddPCR detection system for simultaneous,rapid and absolute quantitative detection of genomic copy numbers of Salmonella typhi,Staphylococcus aureus and L.monocytogenes in the same reaction system was constructed.The multiplex ddPCR detection system could obtain fluorescent amplification with 8(23)distinct clusters under the optimal condition obtained by means of annealing temperature optimization,and the quantitative linear range of these 3 pathogens were 235~0.23、284~0.18、380~0.42 copies/µL,which were almost the same as simplex ddPCR for single strain,and the linear correlation coefficient R^(2) in the quantitative detection range were all greater than 0.999 with excellent repeatability.The constructed multiplex ddPCR detection system had strong specificity,good stability and wide quantitative range,which could provide technical support for absolute quantitative detectio

关 键 词：食源性致病菌 伤寒沙门氏菌 金黄色葡萄球菌 单核细胞增生李斯特氏菌 多重微滴数字PCR (ddPCR) 绝对定量检测 

分 类 号：S3[农业科学—农艺学]