Establishment of Droplet Digital PCR Assay for Quantitative Detection of Pseudomonas cocovenenans in Foods

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

Abstract

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

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.

Figures/Tables 9

References 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