食品中椰毒假单胞菌微滴式数字PCR定量检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2022-0254

摘要/Abstract

摘要：

椰毒假单胞菌可污染包括发酵米面等多种食物，其产生的两种毒素导致严重的食物中毒，威胁人体健康。基于数字PCR技术，建立了针对椰毒假单胞菌的定量测量方法。该方法具有较好的特异性与重复性，人工污染糯米汤样品中的检出限为361 CFU/mL。与平板培养法相比，基于数字PCR技术的测量方法缩短了测量时间，有利于椰毒假单胞菌的快速准确测量，未来可在食品安全监测与中毒分析等领域进行应用。

关键词: 椰毒假单胞菌, 食物中毒, 数字PCR, 平板培养法, 定量测量

Abstract:

Pseudomonas cocovenenans contaminates a variety of foods including fermented rice and noodles. It produces two toxins that cause severe food poisoning, which threatens human health. Based on digital PCR technology, the quantitative method targeting to P. cocovenenans was established. It demonstrated good specificity and repeatability. The detection limit in polluted glutinous rice soup was 361 CFU/mL. Compared with plate counting method, the detection time was shortened obviously via the digital PCR quantitative method, which was conducive to the rapid and accurate measurement of P. cocovenenans. Thus, it may be applied in the fields of food safety monitoring and poisoning analysis in the future.

Key words: Pseudomonas cocovenenans, food poisoning, digital PCR, plate counting, quantitative detection

李会杰, 董莲华, 陈桂芳, 刘思渊, 杨佳怡, 杨靖亚. 食品中椰毒假单胞菌微滴式数字PCR定量检测方法的建立[J]. 生物技术通报, 2023, 39(1): 127-136.

LI Hui-jie, DONG Lian-hua, CHEN Gui-fang, LIU Si-yuan, YANG Jia-yi, YANG Jing-ya. Establishment of Droplet Digital PCR Assay for Quantitative Detection of Pseudomonas cocovenenans in Foods[J]. Biotechnology Bulletin, 2023, 39(1): 127-136.

图/表 9

参考文献 34

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菌株编号No.	菌株名称Name	革兰氏属性Gram property	培养基Culture medium
CICC10574	椰毒假单胞菌Pseudomonas cocovenenans	阴性Negative	LB培养基LB medium
CICC10384	金黄色葡萄球菌Staphylococcus aureus	阳性Positive	营养肉汤培养基Nutrient broth medium
CICC21535	宋内氏志贺氏菌Shigella sonnei	阴性Negative	营养肉汤培养基Nutrient broth medium
CICC10389	大肠埃希氏菌Escherichia coli	阳性Positive	营养肉汤培养基Nutrient broth medium
ATCC8400	沙门氏菌Salmonella	阴性Negative	营养肉汤培养基Nutrient broth medium
CICC21633	单核增生性李斯特氏菌Listeria monocytogenes	阴性Negative	营养肉汤培养基Nutrient broth medium
ATCC6633	枯草芽孢杆菌Bacillus subtilis	阳性Positive	营养肉汤培养基Nutrient broth medium
CICC2219	黄曲霉Aspergillus flavus	阴性Negative	5 °Bé麦芽汁琼脂5 °Bé wort agar medium

菌株编号No.	菌株名称Name	革兰氏属性Gram property	培养基Culture medium
CICC10574	椰毒假单胞菌Pseudomonas cocovenenans	阴性Negative	LB培养基LB medium
CICC10384	金黄色葡萄球菌Staphylococcus aureus	阳性Positive	营养肉汤培养基Nutrient broth medium
CICC21535	宋内氏志贺氏菌Shigella sonnei	阴性Negative	营养肉汤培养基Nutrient broth medium
CICC10389	大肠埃希氏菌Escherichia coli	阳性Positive	营养肉汤培养基Nutrient broth medium
ATCC8400	沙门氏菌Salmonella	阴性Negative	营养肉汤培养基Nutrient broth medium
CICC21633	单核增生性李斯特氏菌Listeria monocytogenes	阴性Negative	营养肉汤培养基Nutrient broth medium
ATCC6633	枯草芽孢杆菌Bacillus subtilis	阳性Positive	营养肉汤培养基Nutrient broth medium
CICC2219	黄曲霉Aspergillus flavus	阴性Negative	5 °Bé麦芽汁琼脂5 °Bé wort agar medium

名称Name	序列Sequence（5'-3'）	长度Length/bp
上游引物Forward primer	TTGTCTGGCGGTAGAAACC	19
下游引物Reward primer	CGAGTCAACTGACCCGAAC	19
探针Probe	FAM-TCGATCCCGTCTGCCT CCACCAATCT-BHQ	26

名称Name	序列Sequence（5'-3'）	长度Length/bp
上游引物Forward primer	TTGTCTGGCGGTAGAAACC	19
下游引物Reward primer	CGAGTCAACTGACCCGAAC	19
探针Probe	FAM-TCGATCCCGTCTGCCT CCACCAATCT-BHQ	26

稀释度Dilution	ddPCR检测结果Results of ddPCR/（CFU·mL^-1）				RSD/%	原菌液浓度Concentration of the original bacterium/（CFU·mL^-1）	1g（CFU·mL^-1）
稀释度Dilution	重复1 Repetition 1	重复2 Repetition 2	重复3 Repetition 3	平均值 Mean	RSD/%	原菌液浓度Concentration of the original bacterium/（CFU·mL^-1）	1g（CFU·mL^-1）
10³	418 941	411 647	409 882	413 490	1.16	4.13×10⁸	8.62
10⁴	45 176	43 764	47 058	45 333	3.65	4.53×10⁸	8.66
10⁵	4 541	4 118	4 329	4 329	4.89	4.33×10⁸	8.64
10⁶	1 518	1 459	1 624	1 533	5.44	1.53×10⁹	9.18
10⁷	329	376	271	325	16.29	3.25×1⁰9	9.51