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Multiple-PCR rapid detection method of salmonella and escherichia coli

A technology for the detection of Escherichia coli and Escherichia coli O78, which is applied in the field of multiplex PCR rapid detection of Salmonella and Escherichia coli O78, can solve the problems of specificity, low sensitivity, unpredictable interference of primers and probes, cumbersome and complicated operation, etc., and achieve high detection Sensitivity and specificity, shortening the detection time, and improving the detection efficiency

Active Publication Date: 2015-06-17
青岛智测检验检测有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[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. are low and cannot meet the needs of rapid detection
The fluorescent PCR method introduced by Applied Biosystems of the United States in 1996 quickly developed into an important means of molecular biology detection due to its advantages of low pollution and high sensitivity. Interference is difficult to predict, and fluorescent probes are expensive, so it is difficult to establish a multiplex fluorescent PCR method, and its practicability is greatly affected

Method used

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  • Multiple-PCR rapid detection method of salmonella and escherichia coli
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  • Multiple-PCR rapid detection method of salmonella and escherichia coli

Examples

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Embodiment 1

[0033] A multiplex PCR rapid detection method for Salmonella and Escherichia coli O78, using multiplex PCR technology to amplify the specific gene of Salmonella and Escherichia coli O78 and the partial sequence of bacterial 16s rDNA, and then detect the PCR amplification by agarose gel electrophoresis product.

[0034] 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.

[0035] Main reagent used in this embodiment:

[0036] Taq DNA polymerase, dNTPs, 2000bp DNA Marker [Bao Biological Engineering (Dalian) Co., Ltd.], primers (Beijing Huada Gene Technology Co., Ltd.), LB (Qingdao Haibo Biotech...

Embodiment 2

[0061] A multiplex PCR rapid detection method for Salmonella and Escherichia coli O78 in a chicken manure sample, adding Salmonella or Escherichia coli O78 to the aseptic chicken manure for enrichment culture, extracting the total DNA in the chicken manure sample, using the method described in Example 1 The above system was used for multiplex PCR amplification, and then the PCR amplification products were detected by agarose gel electrophoresis.

[0062] The main reagents and instruments used in this embodiment are the same as in Example 1.

[0063] The multiple PCR rapid detection method for Salmonella and Escherichia coli O78 in the above-mentioned chicken manure sample specifically comprises the following steps:

[0064] 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; centrif...

Embodiment 3

[0068] A multiplex PCR rapid detection method for Salmonella and Escherichia coli O78 in milk powder samples, adding Salmonella or Escherichia coli O78 to aseptic milk powder and then enriching culture, extracting the total DNA in the milk powder samples, using the system described in Example 1 to carry out Multiplex PCR amplification followed by detection of PCR amplification products by agarose gel electrophoresis.

[0069] The main reagents and instruments used in this embodiment are the same as in Example 1.

[0070] The multiple PCR rapid detection method for Salmonella and Escherichia coli O78 in the above-mentioned milk powder sample specifically comprises the following steps:

[0071] 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 supernatant , add 100 μL ddH 2...

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Abstract

The invention discloses a multiple-PCR rapid detection method of salmonella and escherichia coli. A primer for performing multiple-PCR rapid detection on salmonella and escherichia coli O78 comprises (1) a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; or (2) a nucleotide sequence having the basic sequence homology with the nucleotide sequence in (1) and having the function as same as that of the nucleotide sequence in (1). According to the detection method, specific genes of salmonella and escherichia coli O78 and a part of sequences of bacteria 16s rDNA are amplified once by using the multiple-PCR technology, and a PCR amplification product is detected by means of agarose gel electrophoresis. According to the method, the problems of long period (4-7 days) of a traditional pathogenic bacteria detection technology, tedious and complicated operations and relatively-low specificity and sensitivity are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multiple 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. The fluorescent PCR method introduced by Applied Biosystems of the United States in 1996 quickly developed into an important means of molecular biology detection d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42C12R1/19
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 胡茂秀王莎莎俞超
Owner 青岛智测检验检测有限公司
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