三重微滴数字PCR定量检测即食米粉中致病菌方法研究
作者:
作者单位:

1.广州市食品检验所,广东 广州 511400;2.广州知识产权保护中心,广东 广州 510010;3.澳实分析检测有限公司(加拿大),加拿大 不列颠哥伦比亚省 V4A1W9

作者简介:

孙雪奇 女 主任药师 研究方向为食品安全质量研究控制 E-mail:sun-xueqi@163.com

通讯作者:

肖剑 男 高级工程师 研究方向为食品安全质量研究控制 E-mail:xjhq521@163.com

中图分类号:

R155

基金项目:

广州市科技计划项目(201904010253);广州市科技计划项目(202206010095)


Research on the quantification method to detect pathogenic bacteria in instant rice noodles by 3-plex droplet digital polymerase chain reaction test
Author:
Affiliation:

1.Guangzhou Institute for Food Inspection, Guangdong Guangzhou 511400, China;2.Guangzhou Intellectual Property Protection Center, Guangdong Guangzhou 510010, China;3.ALS Canada Ltd., British Columbia V4A1W9, Canada

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    摘要:

    目的 建立三重微滴数字PCR(ddPCR)方法同时定量检测即食食品中的沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌。方法 选择以沙门菌ttrA/ttrC、蜡样芽胞杆菌essC、单核细胞增生李斯特菌侵袭相关内肽酶基因等3个单拷贝基因对应的3对引物探针,采用实时荧光定量PCR验证引物/探针特异性后,建立三重ddPCR方法同时定量检测3种致病菌的拷贝数。结果 该方法的线性范围分别为:沙门菌25~22 687 copies/20 μL;蜡样芽胞杆菌19~15 620 copies/20 μL;单核细胞增生李斯特菌18~23 373 copies/20 μL,线性相关因子r2≥0.999,6个浓度3次重复测定3种菌的相对标准偏差(RSD)≤12%,重复性好,对于上述菌株的最低检出限分别为6、3和7 copies/20 μL;采用已建立的ddPCR方法和平板计数方法对模拟染菌米粉样品进行检测,两种方法测定值结果RSD小于9%,结果一致性较好。结论 本研究建立的三重ddPCR同时定量检测即食食品中沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌的方法与平板计数法相比,更快速、灵敏,结果准确。

    Abstract:

    Objective This study aimed to establish a quantitative 3-plex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting the copy numbers of SalmonellaBacillus cereus, and Listeria monocytogenes in instant food.Methods Three pairs of primers and probes corresponding to three single-copy-genes were selected as target genes. The genes were the essC gene in Bacillus cereusttrA/ttrC gene in Salmonella, and invasion-associated endopeptidase gene in Listeria monocytogenes. The specificity of the primers and probes were verified by real-time fluorescence quantitative PCR separately. A 3-plex ddPCR method was constructed to detect the copy numbers of three pathogenic bacteria simultaneously.Results The linear ranges were: 25-22 687 copies/20 μL for Salmonella, 19-15 620 copies/20 μL for Bacillus cereus, and 18-23 373 copies/20 μL for Listeria monocytogenes. The three linear correlation coefficients were r≥0.999. relative standard deviation (RSD)≤12% at six concentrations and repeated thrice, indicating favorable repeatability. The minimum detection limits were six copies/20 μL for Salmonella, three copies/20 μL for Bacillus cereus, and seven copies/20 μL for Listeria monocytogenes. When a simulated sample of contaminated rice noodles was detected by 3-plex ddPCR and the plate counting method, the deviation between these two methods was <9%, indicating a good consistency in the results.Conclusion The 3-plex ddPCR method for the simultaneous and quantitative detection of Salmonella, Bacillus cereus, and Listeria monocytogenes in instant food was quicker, more sensitive, and more accurate than the plate-counting method.

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孙雪奇,尹玮璐,蒋佳希,张明明,梁美丹,曾善美,肖剑.三重微滴数字PCR定量检测即食米粉中致病菌方法研究[J].中国食品卫生杂志,2023,35(11):1571-1578.

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  • 收稿日期:2022-12-02
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  • 在线发布日期: 2024-02-20
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