Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (1): 127-136.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0254
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LI Hui-jie1,2(), DONG Lian-hua2, CHEN Gui-fang3, LIU Si-yuan2, YANG Jia-yi2(), YANG Jing-ya1()
Received:
2022-03-01
Online:
2023-01-26
Published:
2023-02-02
Contact:
YANG Jia-yi,YANG Jing-ya
E-mail:15713680318@163.com;yangjy2018@nim.ac.cn;jyyang@shou.edu.cn
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.
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 |
Table 1 Strains for experiment
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 |
Table 2 Primers’ and probes’ sequences of ddPCR
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 |
Fig. 2 Specificity of ddPCR for Pseudomonas cocovenenans 1:Pseudomonas cocovenenans,CICC10574;2:Staphylococcus aureus,CICC10384;3:Shigella sonnei,CICC21535;4:Escherichia coli,CICC10389;5:Salmonella,ATCC8400;6:Listeria monocytogenes,CICC21633;7:Bacillus subtilis,ATCC6633;8:Aspergillus flavus,CICC2219
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) | |||
---|---|---|---|---|---|---|---|
重复1 Repetition 1 | 重复2 Repetition 2 | 重复3 Repetition 3 | 平均值 Mean | ||||
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 |
Table 3 ddPCR results of gradient dilutions of P. cocovenenans
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) | |||
---|---|---|---|---|---|---|---|
重复1 Repetition 1 | 重复2 Repetition 2 | 重复3 Repetition 3 | 平均值 Mean | ||||
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 |
稀释度 Dilution | qPCR检测结果Results of qPCR/(CFU·mL-1) | RSD/% | 原菌液浓度Concentration of the original bacterium/(CFU·mL-1) | 1g(CFU·mL-1) | |||
---|---|---|---|---|---|---|---|
重复1 Repetition 1 | 重复2 Repetition 2 | 重复3 Repetition 3 | 平均值 Mean | ||||
101 | 271 607 078 | 283 669 787 | 271 607 078 | 275 627 980 | 2.53 | 2.75×109 | 9.44 |
102 | 12 112 815 | 12 729 551 | 11 815 744 | 12 219 370 | 3.81 | 1.22×109 | 9.08 |
103 | 351 986 | 349 807 | 367 618 | 356 470 | 2.73 | 3.56×108 | 8.55 |
104 | 42 647 | 42 647 | 39 585 | 41 626 | 4.25 | 4.16×108 | 8.62 |
105 | 5 567 | 5 103 | 5 601 | 5 424 | 5.13 | 5.42×108 | 8.73 |
Table 4 qPCR results of gradient dilutions of P. cocovenenans
稀释度 Dilution | qPCR检测结果Results of qPCR/(CFU·mL-1) | RSD/% | 原菌液浓度Concentration of the original bacterium/(CFU·mL-1) | 1g(CFU·mL-1) | |||
---|---|---|---|---|---|---|---|
重复1 Repetition 1 | 重复2 Repetition 2 | 重复3 Repetition 3 | 平均值 Mean | ||||
101 | 271 607 078 | 283 669 787 | 271 607 078 | 275 627 980 | 2.53 | 2.75×109 | 9.44 |
102 | 12 112 815 | 12 729 551 | 11 815 744 | 12 219 370 | 3.81 | 1.22×109 | 9.08 |
103 | 351 986 | 349 807 | 367 618 | 356 470 | 2.73 | 3.56×108 | 8.55 |
104 | 42 647 | 42 647 | 39 585 | 41 626 | 4.25 | 4.16×108 | 8.62 |
105 | 5 567 | 5 103 | 5 601 | 5 424 | 5.13 | 5.42×108 | 8.73 |
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