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[Objective] To establish a dual detection method for contaminations by six foodborne pathogens (Cronobacter, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Salmonella, and Listeria monocytogenes) in formula milk powder in a rapid manner. [Methods] Enzymatic recombinase amplification (ERA) is a novel isothermal amplification technology that exponentially amplifies trace amounts of DNA or RNA in 10-30 min at 25-42 ℃. The primers and probe of ERA for the detection of Cronobacter were designed. Meanwhile, the ERA primers and probes suitable for the detection of E. coli O157:H7, B. cereus, S. aureus, Salmonella, and L. monocytogenes were screened. Further, through pairwise combination and cross-reactivity analysis, as well as method optimization, the dual ERA detection system was established. The limit of detection and accuracy of the method were determined by application of this method in the detection of simulated contaminations and actual samples. [Results] Three groups of dual ERA systems were established, achieving the detection of six pathogens in 16 min 10 s. The established method showed the sensitivity of 1 ng/μL in the DNA detection of the combinations of Cronobacter with E. coli O157:H7, B. cereus, and S. aureus, while it showed the sensitivity of 10-1 ng/μL in the DNA detection of Salmonella and L. monocytogenes. The results of the simulation contaminations showed that the limit of detection of the method was 1 CFU/mL. The dual ERA method established in this study was then adopted to detect 37 commercially available formula milk powder samples near the expiration date. The detection rates of B. cereus and L. monocytogenes were 37.84% and 21.62%, respectively. The results were consistent with those of the real-time PCR (industry standard method), confirming the accuracy of the dual ERA method established in this study. [Conclusion] The dual ERA method established in this study exhibits high specificity and high sensitivity. Moreover, it takes merely approximately 25 min from DNA extraction to obtaining the detection results, and it is capable of simultaneously detecting six pathogens, demonstrating high efficiency. This method is of importance for the rapid screening of foodborne 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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