Method for simultaneously detecting various pathogenic bacteria in raw milk and detection kit
A technology of pathogenic bacteria and raw milk, applied in the field of detection of bacteria in raw milk, can solve the problems of inability to detect multiple pathogenic bacteria at the same time, unsuitable detection of milk, and long time consumption, so as to save time for bacterial enrichment and save Steps and time, the effect of simple operation
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specific Embodiment approach 1
[0013] Specific embodiment 1: In this embodiment 1, 200 mL of raw milk to be tested is centrifuged with a centrifugal force of 3000 g for 15 min to remove the fat in the upper layer to obtain a fat-free sample, and 30 mL of EDTA- 2Na solution, incubate in a water bath at 37°C for 30min; 2. Filter the sample after step 1 incubating through a Chua filter, cut the filter membrane into pieces, put it into a dry and sterile flask, and add 20mL 3. Collect the eluate and centrifuge at a centrifugal force of 10,000g for 5 minutes, remove the lower layer of bacterial sediment and resuspend it with 1mL of sterile double-distilled aqueous solution, and then use a centrifugal force of 10,000g Centrifuge for 5 minutes, discard the supernatant, and obtain the thallus; 4. Utilize the bacterial genome kit to extract bacterial genomic DNA as a multiplex PCR template, and finally perform multiplex PCR, and then identify the PCR amplification products with 2% gel electrophoresis, and use Gel ima...
specific Embodiment approach 2
[0015] Specific embodiment 2: In the kit for simultaneously detecting multiple pathogenic bacteria in this embodiment, 1 part of Taq enzyme solution with a concentration of 2.5U / μL, 20 parts of multiplex PCR reaction solution, and 1 part of negative The control solution and 1 positive control solution consist of 5 μL of 10×PCR buffer, 3 μL of 10 -2 mol / L dNTPs, 8 μL primer solution and 28 μL sterilized double distilled water, the primer solution contains 2.5×10 Listeria monocytogenes primers -11 mol, Salmonella primer 1.25×10 -11 mol, Staphylococcus aureus primer 2.5×10 -11 mol, Escherichia coli O157: H7 primer 1.25×10 -11 mol and Bacillus cereus primers 2.5 x 10 -11 mol; the positive control solution is the genomic DNA extracted from the culture solution of five standard strains of pathogenic bacteria; the negative control solution is sterilized double distilled water.
[0016] The kit of this embodiment is used for the multiplex PCR detection in step 4 of the specific em...
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