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Objective To establish an automatic, fast and high-resolution detection method for 5 kinds of common pathogenic bacteria in aquatic products, and improve the efficiency and accuracy of detecting pathogenic microorganisms in aquatic products. Methods Genes of owpW, tlh, invA, femA, and prfA from Vibrio cholera, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, and Listeria monocytogenes were amplified by multiple polymerase chain reaction (PCR). PCR products were used as templates for single nucleotide extention and molecular weight of the extended probes was detected on the mass spectrometer. The molecular weight of probes for genes owpW、tlh、invA、femA and prfA were 4848, 5435, 5890, 6560 and 7096 Da. The molecular weights of the extended probes were 5119 Da (plus A), 5697 Da (plus T), 6137 Da (plus C), 6822 Da (plus T) and 7383 Da (plus G), respectively. This finally determined system was verified by reproducibility test, specificity test, sensitivity test and detection test of artificially contaminated aquatic samples. Results Using a sample of mixed DNA from 5 kinds of different bacteria as a template for nucleic acid mass spectrometry detection, the corresponding 5 kinds of probes could be extended simultaneously with an extension efficiency greater than 80%. The above 5 kinds of bacteria would not be detected in samples using interfering bacteria as templates. The sensitivity for detecting Salmonella, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, and Vibrio cholerae could reach 150, 350, 160, 130, and 180 CFU/mL, respectively. Conclusion This method demonstrates good reproducibility, specificity, and sensitivity, and has a high degree of automation, which can meet the detection needs of the above 5 kinds of microorganisms in aquatic products simultaneously.
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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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