Primer group for detecting escherichia coli O157 in feed and detection method of primer group

A technology for detection of Escherichia coli and its detection method, which is applied in the detection of Escherichia coli O157 in feed and its detection field, which can solve the problems of low sensitivity and achieve the effect of improving sensitivity and detection sensitivity

Pending Publication Date: 2017-12-19
重庆市动物疫病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a primer set for detecting Escherichia coli O157 in feed based on the loop-mediated isothermal amplification technique. Another purpose of the present invention is to use the primer set to detect A detection method for E. coli O157 in feed to solve the technical problem of low sensitivity of existing loop-mediated isothermal amplification technology in the detection of E. coli O157 in feed

Method used

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  • Primer group for detecting escherichia coli O157 in feed and detection method of primer group
  • Primer group for detecting escherichia coli O157 in feed and detection method of primer group
  • Primer group for detecting escherichia coli O157 in feed and detection method of primer group

Examples

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Effect test

Embodiment 1

[0068] Escherichia coli O157:H7 detection in feed, the detection process is as follows Figure 10 As shown, specifically:

[0069] Escherichia coli O157:H7 cultivation: First, mix 500g of concentrated feed confirmed to be free of Escherichia coli O157:H7 with 500g of pig bone meal. Weigh 8 samples of 25g and artificially introduce Escherichia coli O157:H7, the introduction amount is 2CFU / 25g, 4CFU / 25g, 6CFU / 25g, 8CFU / 25g, 10CFU / 25g, 12CFU / 25g, 14CFU / 25g, and then introduce into the large intestine The samples of Bacillus O157:H7 were added into sterile homogeneous bags filled with 225mL modified EC broth (purchased from Guangdong Huankai), and placed in a constant temperature incubator at 36°C±1°C for 18h to obtain the enrichment solution;

[0070] DNA extraction: Take 7000g of the aforementioned 1mL enrichment solution and centrifuge it for 1min, remove the supernatant, then wash the precipitate twice with double-distilled water, suspend the precipitate with 100μL double-dis...

Embodiment 2

[0075] Cultivate Escherichia coli O157:H7 with reference to the steps in Example 1 to obtain the enrichment solution, and perform ordinary PCR amplification test according to the following method.

[0076] Ordinary PCR amplification reaction: Add 10 μL of 5×taq DNA polymerase buffer, 1 μL of dNTP (deoxyribonucleoside triphosphate) mixture, MgCl 2 6 μL, 2 μL of internal primer (FIP), 2 μL of internal primer (BIP), 2 μL of external primer (F3), 2 μL of external primer (B3), 2 μL of template DNA, 0.25 μL of taq DNA polymerase, and add the balance to 50 μL with distilled water to obtain the reaction system. Place the PCR tube in the PCR machine and perform the reaction according to the following conditions: denature at 95°C for 2 minutes; then 95°C for 30s; 57°C for 30s; 72°C for 30s, and so on for 35 cycles; then extend at 72°C for 5 minutes. After the reaction, observe the results by 2% agarose gel electrophoresis, the results are shown in Figure 11 , the product should be 2...

Embodiment 3-6

[0079] Referring to Example 1, detection of Escherichia coli O157:H7 in feed. Wherein, the concentration of each component of the loop-mediated isothermal amplification reaction system is selected according to Table 4;

[0080] Each component concentration of reaction system in table 4 embodiment 3-6

[0081]

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Abstract

The invention provides a primer group for detecting escherichia coli O157 in feed. The primer group comprises an inner primer FIP, an inner primer BIP, an outer primer F3, an outer primer B3 and a loop primer LB. A method of detecting the escherichia coli O157 in the feed by using the primer group comprises the steps of escherichia coli O157 culture, DNA (deoxyribonucleic acid) extraction, loop-mediated isothermal amplification reaction and result observation. Tests show that the primer group has no specific expression for enterobacter aerogenes, escherichia coli and salmonella enteritidis, and obvious expression and high specificity for the escherichia coli O157; the feed is detected by a loop-mediated isothermal amplification detection method; a detection result is very precise; and a detection limit of the escherichia coli O157 in the feed is 2 CFU (colony forming unit)/25g. In addition, the primer group has the advantages of high detection speed, convenience in operation and the like for the detection of the feed.

Description

technical field [0001] The invention relates to a detection method of enterobacteria, in particular to a primer set for detection of Escherichia coli O157 in feed based on a loop-mediated isothermal amplification technique and a detection method thereof. Background technique [0002] Escherichia coli O157, Escherichia spp., Gram stain negative, kinetic test positive. Escherichia coli O157 is a type of enterobacteria that can cause symptoms such as intestinal hemorrhagic diarrhea and enteritis in humans and animals. Escherichia coli O157 generally spreads the disease through drinking water, food, excrement, close contact, etc. In severe cases, it can be life-threatening. Food hygiene authorities have listed Escherichia coli O157 as a routine detection item. [0003] At present, the detection methods of Escherichia coli O157 include conventional methods (bacterial culture, isolation, identification), gold standard test paper method, monoclonal enzyme-linked immunoassay screen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/19
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 胡健范首君何义刚丁平蔺露张毅欧阳吴莉
Owner 重庆市动物疫病预防控制中心
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