Full
Outline
To establish a multiplex digital polymerase chain reaction (dPCR) quantitative detection system for Cronobacter (Escherichia sakazakii), Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella, Listeria monocytogenes and pathogenic Escherichia coli O157.
Species-specific genes were screened from the genomes of the 7 kinds of pathogenic bacteria in the National Center for Biotechnology Information (NCBI) genome database, and their conservation was analyzed. Primer-probes were designed based on the conserved regions. The primer specificity and probe structure were analyzed by using MFE Primer (V3.1) to alignment against the human genome, bacterial and viral genomes in the NCBI ftp database, followed by the synthesis of primers probes and pathogenic bacterial plasmids. A multiplex dPCR detection system for foodborne pathogens was established by using multicolor coding technology to encode the detection targets of the 7 kinds of pathogenic bacteria with different fluorescence channel combination modes, and the specificity, accuracy, linearity and repeatability of the system were verified.
The detection system was characterized by high specificity, accuracy and reproducibility, with linear ranges of 15.55–17336.02 copies/μL for Cronobacter (Escherichia sakazakii), 11.92–12755.55 copies/μL for Staphylococcus aureus, 9.47–11811.35 copies/μL for Vibrio cholerae, 9.86–11631.06 copies/μL for Vibrio parahaemolyticus, 12.20–14244.65 copies/μL for Salmonella, 16.70–15329.10 copies/μL for Listeria monocytogenes, and 28.22–26325.95 copies/μL for pathogenic Escherichia coli O157.
The construction of a multiplex dPCR detection system for various foodborne pathogens, utilizing multicolor coding technology, facilitate the simultaneous detection of 7 kinds of foodborne pathogens. This system offer a valuable technical reference for pathogen surveillance, contamination source tracing and emergency response in the fields of food, medicine, and the environment, demonstrating broad application potential. It will facilitate further exploration of a super-multiplex dPCR detection system for pathogens.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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