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Listeria monocytogenes ic-pcr detection kit and preparation method

A monocytogenes, IC-PCR technology, applied in the field of routine detection of Listeria monocytogenes in food, can solve the problems of increasing detection time, difficult to meet rapid detection, etc., achieve sensitive capture and reduce detection sensitivity Falling, high-sensitivity effects

Inactive Publication Date: 2011-12-21
刘箐 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used enrichment method is biological culture. Although this method can greatly improve the detection rate and detection sensitivity, it increases the detection time and is difficult to meet the requirements of rapid detection.

Method used

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  • Listeria monocytogenes ic-pcr detection kit and preparation method
  • Listeria monocytogenes ic-pcr detection kit and preparation method
  • Listeria monocytogenes ic-pcr detection kit and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: An IC-PCR detection kit for Listeria monocytogenes, comprising a PCR tube coated with LM monoclonal antibody, containing Mg 2+ 1 tube of PCR Buffer (10×), 1 tube of 2.5mM dNTP, 1 tube of 5U Taq DNA polymerase, 1 tube of 10 pmol / μL primers 5’-CGGAGGTTCCGCAAAAGATG-3’ and 5’-CCTCCAGAGTGATCGATGTT-3’, sterilized ddH 2 1 tube of O, 1 tube of positive control, 1 tube of negative control, and 1 tube of PBST washing buffer.

Embodiment 2

[0038] Example 2: see figure 2 , a method for using the IC-PCR detection kit of Listeria monocytogenes, its main feature is that the steps are:

[0039] (1) Purification and enrichment of pathogenic bacteria: add the sample to be tested, positive control (heat-inactivated Listeria monocytogenes), negative control (sterile PBS) into the PCR tube coated with LM monoclonal antibody ), incubate at 37°C for 4 hours; then wash the PCR tube 3 times with PBST washing buffer, 1 min each time, and discard the washing solution;

[0040] (2) PCR amplification: Add PCR master solution system (10×PCR buffer 5μL, 2.5mM dNTP 2μL, Taq DNA Polymerase 2.5 U, primer 0.6pmol / μL, ddH 2 O to make up 50 μL); perform PCR amplification under the conditions of pre-denaturation at 95°C for 20 min, 35 cycles at 95°C for 30 s, 55°C for 45 s, and 62°C for 30 s, extension at 72°C for 5 min, and storage at 4°C;

[0041] (3) Result analysis: Take 10 μL of the amplification product to detect the target band ...

Embodiment 3

[0042] Embodiment 3: a kind of preparation method of the IC-PCR detection kit specific PCR coating tube of Listeria monocytogenes, it is characterized in that the steps are:

[0043] (1) Dilute the LM monoclonal antibody to 5.0 μg / mL with coating buffer, and add 50 μL of the diluted antibody to the PCR tube to coat the antibody overnight at 4°C;

[0044] (2) Wash the PCR tube 3 times with PBST, 1 min each time; dry the water in the PCR tube, and store at -20°C;

[0045] (3) Coating buffer formula: anhydrous sodium carbonate 1.59g / L, sodium bicarbonate 2.93g / L, sodium azide 0.2g / L, adjust pH to 9.6; PBST washing buffer formula: sodium chloride 8g / L, anhydrous disodium hydrogen phosphate 1.15g / L, anhydrous potassium dihydrogen phosphate 0.2g / L, potassium chloride 0.2g / L, Tween 20 0.5ml / L, adjust the pH to 7.4.

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Abstract

The invention relates to an immunocapture PCR (Immuno-capturePCR, IC-PCR) detection kit for one of the four major food-borne pathogenic bacteria at the molecular level of Listeria monocytogenes (Listeriamonocytogenes, LM). For the routine detection of Listeria monocytogenes in food. The kit mainly includes: PCR tube coated with LM monoclonal antibody, PCRBuffer (10×) tube containing Mg2+, 2.5mMdNTP tube, 5UTaq DNA polymerase tube, 10pmol / μL primer 5′-CGGAGGTTCCGCAAAAGATG-3′ and 5′-CCTCCAGAGTGATCGATGTT 1 tube for each of -3', sterilized ddH2O tube, positive control tube, negative control tube, 1 tube of PBST washing buffer. The advantage of this kit is that it does not need to extract the DNA step, and the test result can be obtained by using the IC-PCR detection kit directly within 7 hours, and the target bacteria can be detected sensitively, specifically and quickly.

Description

technical field [0001] The present invention relates to a method against Listeria monocytogenes (Listeria monocytogenes), one of four major food-borne pathogens Listeria monocytogenes, LM ) molecular level immunocapture PCR (Immuno-capture PCR, IC-PCR) detection kit, which can be used for routine detection of Listeria monocytogenes in food. Background technique [0002] In recent years, food-borne diseases have occurred frequently, and food safety issues have become a public health issue of global concern, and food-borne pathogens are a serious hidden danger of food safety problems. Listeria monocytogenes ( Listeria monocytogenes, LM ) is a common food-borne pathogenic bacteria that widely exists in nature and can infect a variety of meat products, dairy products, aquatic products and plant products; it is the most pathogenic bacterium in the Listeria genus and can cause Severe zoonotic diseases, such as meningitis, sepsis and abortion, etc., pregnant women, newborns, the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 刘雅莉刘箐
Owner 刘箐
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