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To establish and evaluate a real-time fluorescent quantitative PCR (qPCR) method for detecting Salmonella paratyphi B in dairy products.
Specific invA gene of Salmonella paratyphi B was used as the target to design primers. After optimizing the qPCR reaction system and conditions with SYBR GreenⅠ dye, amplification was performed on gradient diluted plasmid standards to establish a standard curve. The sensitivity, specificity and repeatability of the method were systematically verified, and the detection performance was compared with the traditional culture method.
The standard curve equation was y=-3.186 6 +13.172, with a correlation coefficient (R2) of 0.9949. The amplification efficiency (E%) was 105.9%. The linear relationship was good. The qPCR method for detecting Salmonella paratyphi B had a sensitivity of 17.9 copies/μL. In the specificity verification, only Salmonella paratyphi B showed positive amplification, while Escherichia coli, Staphylococcus aureus, etc. were negative. There was no cross-reaction. The repeatability test showed that the intra-group coefficient of variation was 0.2%~0.69%, and the inter-group coefficient of variation was 0.49%~1.54%, all of which were less than 3%, indicating excellent stability. Compared with the traditional culture method (requiring 5~7 days), this qPCR method can shorten the detection time to within 6 hours, significantly improving the detection efficiency.
The SYBR Green Ⅰ qPCR method established in this study has the advantages of high sensitivity, strong specificity, good repeatability, simple operation and rapid detection, providing reliable technical support for the rapid screening, precise quantification and food safety risk prevention and control of Salmonella paratyphi B in dairy products.
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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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