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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.

Inactive Publication Date: 2015-09-30
QINGDAO KANGLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only the concentration of super primers is sufficient for exponential amplification, which overcomes the amplification bias problem of multiplex PCR, improves the detection sensitivity of traditional multiplex PCR by 2-3 orders of magnitude, and solves the problem of false negative results caused by traditional multiplex PCR technology

Method used

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  • Dual Tem-PCR quick detection method for salmonella and escherichia coli O78
  • Dual Tem-PCR quick detection method for salmonella and escherichia coli O78
  • Dual Tem-PCR quick detection method for salmonella and escherichia coli O78

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses a dual Tem-PCR quick detection method for salmonella and escherichia coli O78. According to the dual Tem-PCR quick detection method for the salmonella and the escherichia coli O78, invA gene primer of the salmonella and O-antigen gene primer of the escherichia coli are used for conducting dual Tem-PCR amplification directly on extracted mixing DNA after bacteria enrichment is conducted on a complex sample, and the result is interpreted through gel electrophoresis. A Tem-PCR technology and a dual PCR technology are combined, the problem of enrichment preference of the PCR is solved, the sensitivity degree is improved by 2 to 3 orders of magnitudes compared with an ordinary dual PCR detection, the detection level that the number of sample contaminated bacteria is a single digit can be reached, and the problem that a traditional dual PCR technology is prone to lead to false negative result. According to the dual Tem-PCR quick detection method for the salmonella and the escherichia coli O78, the salmonella and the escherichia coli O78 can be detected quickly, conveniently, excellently and sensitively, wide-range popularization is easy, the salmonella and the escherichia coli O78 can be used for bacteria identification, disease diagnosis and epidemiological investigation, and wide market prospect and comparatively large economic benefits are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double Tem-PCR rapid detection method and application of Salmonella and Escherichia coli O78. Background technique [0002] Rapid detection and identification of pathogenic bacteria is the prerequisite for timely and effective prevention and control of the spread of pathogenic bacteria. It is of great significance to the healthy and normal development of the livestock and poultry industry, food inspection and quarantine safety, and human health. [0003] At present, pathogenic bacteria in food sanitation microbiology inspection (GB / T 4789.1-2003) still mainly rely on traditional bacterial culture, serology, biochemical identification and other methods. Both are low and cannot meet the needs of rapid detection. With the development of nucleic acid-based molecular microorganism detection technology, especially the application of polymerase chain reaction (Polymerase Chai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/6848C12Q2537/143Y02A50/30
Inventor 俞超胡茂秀王莎莎
Owner QINGDAO KANGLAND BIOTECH
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