Dual Tem-PCR quick detection method for salmonella and escherichia coli O78
A technology for the detection of Escherichia coli, which is applied in the field of double Tem-PCR rapid detection of Salmonella and Escherichia coli O78, can solve the problems of false negatives, achieve strong specificity and sensitivity, simple operation, and shorten the detection time.
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Embodiment 1
[0040] A double Tem-PCR rapid detection method for Salmonella and Escherichia coli O78 uses double Tem-PCR technology to amplify the specific genes of Salmonella and Escherichia coli O78 at one time, and then detects the PCR amplification products by agarose gel electrophoresis.
[0041] The pathogenic bacteria used in this example were purchased from China Institute of Industrial Microbiology, China Veterinary Culture Collection Center and Guangzhou Institute of Microbiology Culture Collection Center: Salmonella enterica (Salmonella enterica): CICC21510, Salmonella typhimurium (S.typhimurium): CMCC50115, Salmonella choleraesuis (S.choleraesuis): ATCC13312; Escherichia coli O78 (Escherichia coli O78): CVCC1490, ATCC35401.
[0042] Main reagent used in this embodiment:
[0043] Taq DNA polymerase, dNTPs, 1000bp DNA Marker [Bao Biological Engineering (Dalian) Co., Ltd.], primers (Beijing Huada Gene Technology Co., Ltd.), LB (Qingdao Haibo Biotechnology Co., Ltd.).
[0044] Main...
Embodiment 2
[0068] A double Tem-PCR rapid detection method for Salmonella and Escherichia coli O78 in a chicken manure sample, adding Salmonella or Escherichia coli O78 to sterile chicken manure and then enriching culture, extracting the total DNA in the chicken manure sample, using Example 1 Double Tem-PCR amplification was performed with the system described in , and then the PCR amplification products were detected by agarose gel electrophoresis.
[0069] The main reagents and instruments used in this embodiment are the same as in Example 1.
[0070] The double Tem-PCR rapid detection method of Salmonella and Escherichia coli O78 in the above-mentioned chicken manure sample specifically comprises the following steps:
[0071] Take 1g of fresh chicken manure sterilized by high temperature and high pressure, add 10-fold gradient dilution of Salmonella and Escherichia coli O78 bacterial solution 1mL, make up 5mL of LB liquid medium, 37°C, 200rpm, enrich the bacteria for 5h; centrifuge at lo...
Embodiment 3
[0075] A double Tem-PCR rapid detection method for Salmonella and Escherichia coli O78 in a milk powder sample, adding Salmonella or Escherichia coli O78 to aseptic milk powder and then enriching culture, extracting the total DNA in the milk powder sample, using the method described in Example 1 The system was subjected to double Tem-PCR amplification, and then the PCR amplification products were detected by agarose gel electrophoresis.
[0076] The main reagents and instruments used in this embodiment are the same as in Example 1.
[0077] The double Tem-PCR rapid detection method for Salmonella and Escherichia coli O78 in the above-mentioned milk powder sample specifically comprises the following steps:
[0078] Take 5g of milk powder and add it to 100mL LB liquid medium. After autoclaving, take 1mL and add 10 times gradient dilution of Salmonella and Escherichia coli O78 bacterial solution each 100uL, 37℃250rpm for 5h; centrifuge at 12000rpm for 5min, discard the supernatan...
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