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Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof

A high-sensitivity technology for Listeria monocytogenes, applied in the field of high-sensitivity and rapid detection of Listeria monocytogenes, can solve the problems of cumbersome detection process and low sensitivity, and achieve high sensitivity, short time and reliable results

Active Publication Date: 2012-01-18
上海德诺产品检测有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a high-sensitivity and rapid detection method for Listeria monocytogenes and its kit in view of the existing biological detection method's cumbersome detection process and low sensitivity.

Method used

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  • Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof
  • Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof
  • Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of carboxyl-modified DNA-specific nano-magnetic extraction particles

[0057] 25ml 50mg / ml FeSO 4 ·7H 2 O solution and 25ml 50mg / ml FeCl 3 ·6H 2 Dissolve the O solution in 500ml of distilled water, stir mechanically while ultrasonically in a constant temperature water bath at 40°C, add 50ml of 0.1mol / L sodium hydroxide diluted solution dropwise, then let it stand, pour it into a beaker, wash with water and ethanol respectively, and dry at 75°C for 3 hours , Grinding and crushing. Prepare a mixed solution of cyclohexane, TritonX-100 and n-hexanol in a ratio of 4:1:10 by volume, sonicate, mix 100mL of the above solution with 50mg of ground powder, sonicate, take 400μL of 28% concentrated ammonia water dropwise, add normal Ethyl silicate (TEOS) 2ml, react for 24h, let stand for 48h. Use a magnetic separation column to adsorb the product, wash with ethanol, and dry at 90°C for 5 hours. Dissolve 20 mg / ml of the product and 0.5 mL of N-(2-aminoethy...

Embodiment 2D

[0058] Embodiment 2 DNA magnetic separation and extraction

[0059] Materials: Listeria monocytogenes ATCC13932 strain, Salmonella NCTC 6017 strain, Staphylococcus aureus ATCC 29213 strain and the above strains were all purchased and stored in the microbiological laboratory of Shanghai Denuo Product Testing Co., Ltd. Milk powder was provided by Shanghai Denuo Product Testing Co., Ltd. LB1 medium, LB2 medium and ordinary broth medium were provided by Shanghai Denuo Product Testing Co., Ltd.

[0060] Addition detection: Take Listeria monocytogenes ATCC13932 strain to contaminate the milk powder, take 25g samples to culture according to the LB secondary enrichment method, dilute different gradients, as the sample group 1 to be tested, and count the plates at the same time according to GB / T 4789.30. 2010 verified. Take Salmonella NCTC 6017 strain, inoculate 5ml of nutrient broth, cultivate overnight at 37°C at 150r / min, dilute different gradients with sterile double distilled wa...

Embodiment 3

[0062] Example 3 PCR detection of Listeria monocytogenes DNA extract

[0063] Directly take the DNA eluate to be tested prepared in Example 2 as a template, and use primers Hly and Lap to perform multiplex PCR amplification. The PCR amplification uses the Sangon Bioengineering (Shanghai) Co., Ltd. SK2491-PCR amplification kit.

[0064] Hly: CCGCCTGCAAGTCCTAA / ACAGGAAGAACATCGGGT;

[0065] Iap: GATAAAGCCCAAATAGT / GGACTACTGTTGACGCAA.

[0066] PCR system:

[0067]

[0068]

[0069] PCR program:

[0070] 95°C for 1min; 95°C for 30s, 58.7°C for 20s, 72°C for 20s, 30 cycles; 72°C for 5min; store at 4°C.

[0071] Example 4 Amplified product electrophoresis detection

[0072] The PCR product obtained in Example 3 was detected by agarose gel electrophoresis. Hly primers and Iap primers both detect specific bands, which are positive and suspicious positive results. If no band is detected or only the Hly primer detects a band or only the Lap primer detects a band, it is a negati...

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Abstract

The invention discloses a method for rapid detection of listeria monocytogenes with high sensitivity and a kit thereof, and the method comprises the following steps: (1) lysing biological cells to release DNA; (2) mixing the solution containing the biological DNA and magnetic nano-particles which can be bonded with the DNA, wherein the magnetic nano-particles are gamma-Fe2O3 nano-particles coated by carboxyl-modified silica, allowing the DNA to be bonded onto the surfaces of the magnetic nano-particles under the action of isopropanol, collecting the magnetic nano-particles under the action of an external magnetic field to obtain DNA-bonded magnetic nano-particles; (3) adding water, performing magnetic separation, collecting the aqueous solution to obtain the separated biological DNA solution; (4) performing PCR amplification by a specific primer of a target organism with the DNA solution as a template. The whole detection process of the invention only need 2 hours; the detection limit of listeria monocytogenes is about 1 CFU / 25 g (mL); the time required is short; the sensitivity is high; and the results are reliable.

Description

technical field [0001] The invention relates to the field of biological detection, and specifically discloses a method for rapidly detecting Listeria monocytogenes with high sensitivity and a kit thereof. Background technique [0002] In recent years, the rapid detection technology of food-borne pathogenic bacteria is developing rapidly. In February 2009, the "Food Safety Law of the People's Republic of China" was passed by the Seventh Session of the Standing Committee of the Eleventh National People's Congress. The detection of food-borne pathogenic bacteria is to isolate or in-situ detect the pathogenic bacteria in food raw materials and their production, processing, storage and other environments. The methods used for rapid detection of foodborne pathogens include immunology, biochemistry, biophysics, etc. [0003] Listeria monocytogenes is the only Listeria species recognized to cause disease in humans. It is a common soil bacterium that can also cause severe food pois...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C01G49/06B82Y30/00B82Y40/00
Inventor 孟瑾郑小平韩奕奕支援李敏
Owner 上海德诺产品检测有限公司
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