同步检测克罗诺阪崎肠杆菌和李斯特菌双重微滴式数字PCR方法的建立和应用

摘要/Abstract

摘要： 为建立克罗诺阪崎肠杆菌(Cronobacter sakazakii)和李斯特菌(Listeria monocytogenes)双重微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）快速定量检测方法。针对李斯特菌plcB和克罗诺阪崎肠杆菌ompA基因设计特异性引物探针，优化反应参数后对双重ddPCR方法进行了灵敏度、特异性和重复性实验，最后对80份临床样本进行了检测应用。建立的ddPCR方法的最佳退火温度为56℃，最佳引物浓度为600 nmol/L，克罗诺阪崎肠杆菌和李斯特菌探针浓度分别为200 nmol/L和350 nmol/L，特异性试验显示体系与其他常见食源性细菌无交叉反应，特异性强。重复性试验结果显示，批间与批内变异系数均小于16%，具有良好重复性。敏感性试验结果显示，克罗诺杆菌与单增李斯特菌ddPCR最低检测限（Limit of detection, LoD）分别为7.51拷贝/反应和8.02拷贝/反应，显著高于传统荧光定量PCR（qPCR）方法。此研究构建的双重ddPCR检测体系具有特异性强、稳定性好等优点，可为食源性致病菌的无增菌绝对定量检测提供技术支持。

关键词: 克罗诺阪崎肠杆菌, 李斯特菌, 双重微滴式数字PCR, 定量检测

Abstract: To establish a duplex droplet digital PCR (ddPCR) assay for the absolute quantification of Cronobacter sakazakii (C. sakazakii) and Listeria monocytogenes (L. monocytogenes) without prior enrichment, species-specific primers and probes targeting the ompA gene of C. sakazakii and the plcB gene of L. monocytogenes were designed. After systematic optimization of annealing temperature, primer concentrations and probe concentrations, the analytical sensitivity, specificity and repeatability of the duplex ddPCR were evaluated, and the assay was subsequently applied to 80 clinical samples. The optimal reaction conditions were 56 °C annealing temperature, 600 nM each primer, and 200 nmol/L and 350 nmol/L probe for C. sakazakii and L. monocytogenes, respectively. No cross-reactivity was observed with 20 common food-borne bacteria, indicating high specificity. Inter- and intra-assay coefficients of variation were both <16 %, demonstrating excellent repeatability. The limits of detection (LoD) were 7.51 copies/reaction for C. sakazakii and 8.02 copies/reaction for L. monocytogenes—approximately one order of magnitude lower than those of conventional qPCR. The developed duplex ddPCR offers a specific, robust and enrichment-free tool for the absolute quantification of these two important food-borne pathogens.

Key words: Cronobacter sakazakii, Listeria monocytogenes, duplex droplet digital PCR, quantitative detection

中图分类号:

S852.61

孔朋莉韩笑黄康东杨泓莫虹斐帅江冰裘慧. 同步检测克罗诺阪崎肠杆菌和李斯特菌双重微滴式数字PCR方法的建立和应用[J]. 食品科学.

Peng-Li Kong. Establishment and Application of a Duplex Droplet Digital PCR Method for Simultaneous Detection of Cronobacter sakazakii and Listeria monocytogenes[J]. FOOD SCIENCE.

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