Separation and detection method for shewanella putrefaciens in aquatic products
A technology of Shewanella putrefaction and detection method, applied in the biological field, to achieve the effect of strong specificity, high sensitivity and low cost
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Embodiment 1
[0034] Example 1 Detection of Shewanella spoilage bacteria in ice-fresh large yellow croaker during ice storage:
[0035] Aseptically take 10 g of large yellow croaker samples stored in ice for 0, 2, 4, and 6 days, put them into a sterile bag, add 90 mL of 0.1% sterile peptone saline, and beat to mix. After 10-fold dilution of 1ml was taken respectively, take appropriate dilution, take 1ml and smear it on a TSA plate, incubate at 25°C for 48h, pick 30 colonies with different colony shapes, and streak them onto an iron agar plate. Cultivate at 25°C for 48 hours, after black colonies are produced, pick and collect the black colonies, use the Biospin Bacterial DNA Genome Extraction Kit to extract the DNA of the mixed bacteria in the bacterial solution and store them in a 1.5 mL centrifuge tube at -20°C.
[0036] The components of the iron agar plate medium are as follows (the same below): 20 g bacto-peptone, 3 g beef extract, 3 g yeast extract powder, 0.3 g ferric citrate, 0.3 ...
Embodiment 2
[0045] Example 2 Detection of Shewanella putrefaciens in spoiled large yellow croakers:
[0046] Take 25 g of frozen large yellow croaker (about 5 days old) sample, put it into a sterile bag, add 225 mL of 0.1% sterile peptone saline, and beat to mix. After 10-fold dilution of 1ml was taken respectively, take appropriate dilution, take 1ml and smear it on a TSA plate, incubate at 25°C for 48h, pick 30 colonies with different colony shapes, and streak them onto an iron agar plate. After culturing at 25°C for 48 hours, after black colonies were produced, the bacteria were collected by centrifugation, and the DNA of the mixed bacteria in the bacterial solution was extracted using the Biospin Bacterial DNA Genome Extraction Kit and stored in a 1.5 mL centrifuge tube at -20°C.
[0047] Extracted H 2 The DNA of S bacteria was detected by PCR. The total volume of the reaction system was 50 μl. The amount of each reagent added in the PCR reaction system was as follows:
[0048] P...
Embodiment 3
[0055] Example 3 Detection of Shewanella spoilage in fresh and live sea bass during refrigeration:
[0056] Fresh perch were killed with crushed ice, put into sterilization bags, and stored in refrigerators at 4 °C and -2 °C, respectively. Samples were taken every 3 days to determine the changes of Shewanella putrefaciens. Specifically: aseptically take 10 g of perch samples under different refrigeration conditions, put them into a sterile bag, add 90 mL of 0.1% sterile peptone saline, and beat to mix. After 10-fold dilution of 1ml, select the appropriate dilution, take 1ml and spread it on a TSA plate, incubate at 25°C for 48 hours, pick 30 colonies with different colony shapes, and inoculate them on an iron agar plate respectively. Continue to culture at ℃ for 48 hours, after black colonies are produced, collect the bacteria, and use the Biospin Bacterial DNA Genome Extraction Kit to extract the H 2 DNA from S bacteria, stored at -20°C.
[0057] Extracted H 2 The DNA o...
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