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Objective To establish subsequently the real-time quantitative polymerase chain reaction (PCR) method that could quickly and accurately identify Cronobacter sakazakii, and design specific primer probes based on the DNA gyrB subunit gene. Methods The target gene sequences were searched and downloaded from National Center for Biotechnology Information (NCBI), sequence comparison was performed using DNAMAN, and primer probes were designed by Primer Express software. This established real-time quantitative PCR method was validated through specificity tests, absolute sensitivity tests, relative sensitivity tests and anti-interference tests. The 40 common pathogenic bacteria standard strains were selected for specificity validation. Results The results of multi-dimensional specificity validation showed that the method was able to specifically detect Cronobacter sakazakii, and there was no non-specific amplification for other closely related Cronobacter and common pathogenic bacteria in food. DNA detection sensitivity was 0.0100 ng/μL, while relative sensitivity was 103 CFU/mL. The anti-interference experiment results showed that mixing interfering bacteria and their DNA with Cronobacter sakazakii DNA and Cronobacter sakazakii did not significantly affect the detection results, indicating that this method had good anti-interference ability. Conclusion The primer probes designed in this study are specific, rapid, sensitive and anti-interference for the detection of Cronobacter sakazakii in food samples under real-time fluorescence PCR method. It can provide technical support for detection of Cronobacter sakazakii in the future.
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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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