Nucleotide sequence for detecting listeria monocytogenes and detection method and detection kit

A technology for mononucleosis and Listeria, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems that have not been widely used, and achieve simple result determination, strong anti-interference, and sensitivity high effect

Inactive Publication Date: 2015-03-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some rapid detection methods for Listeria monocytogenes, except for the ELISA method used in actual detection

Method used

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  • Nucleotide sequence for detecting listeria monocytogenes and detection method and detection kit
  • Nucleotide sequence for detecting listeria monocytogenes and detection method and detection kit
  • Nucleotide sequence for detecting listeria monocytogenes and detection method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Detection of Listeria monocytogenes standard strain CICC21635

[0030] (1) PCR detection of the standard strain CICC21635 of Listeria monocytogenes was carried out by using a pair of primers Lm16 of the present invention.

[0031] The sequence of the primer pair Lm16 used is as follows:

[0032] Lm16F: 5'-CTGTTCGTCGGTCCGTGGTA-3' (SEQ ID NO.2);

[0033] Lm16R: 5'-CAATGCTCTAATGCGGTGGT-3' (SEQ ID NO. 3).

[0034] The corresponding PCR amplification product size is 1623bp.

[0035] The PCR reaction system used in the present invention is as follows:

[0036] The optimized PCR reaction system includes 10×PCR buffer, 5.0~10.0mM MgCl 2 , 1.0~2.0mM dNTP, 10~20μM primer Lm16F, 10~20μM primer Lm16R, 1~10ng / μL genomic DNA, 0.5~1U TaqDNA polymerase, double distilled water to make up to 25μL; the optimal PCR reaction program ① 94°C, 3 min; ② 94°C, 30s; ③ 60°C, 30s; ④ 72°C, 75s; steps ② to ④ cycled 35 times; ⑤ 72°C, 10min; ⑥ 4°C storage.

[0037] Under the optimal reac...

Embodiment 2

[0055] Example 2: Artificial contamination experiment of Listeria monocytogenes

[0056] The standard strain CICC21635 of Listeria monocytogenes was inoculated in BHI medium and cultivated overnight at 37°C and 180rpm. The cell concentration of L. monocytogenes was calculated by serial dilution and plate counting. Mix 1 mL of bacterial solution with different dilution gradients with 9 mL of aseptic milk to make the inoculum of artificially contaminated milk reach 10 -1 -10 3 cfu / mL, then artificially contaminated milk with different inoculum amounts were added to 90 mL of BHI medium, cultured at 37°C and 180 rpm. From 0h to 12h, samples were taken every two hours and stored at -20°C. Genomic DNA contained in the sample is extracted using the poaching method. PCR was performed using the mentioned genomic DNA as a template. The test results are shown in Table 2. In inoculums as low as 1.63 x 10 -1 cfu / mL Listeria monocytogenes can be detected after 8 hours of cultivation....

Embodiment 3

[0059] Embodiment 3: Use the test kit in the present invention to carry out commercially available sample detection

[0060] According to the best system obtained by optimizing the reaction system in the present invention, the Listeria monocytogenes detection kit is constructed, and based on this, commercially available samples are detected, and a variety of pathogenic bacteria in SN / T 1869-2007 food is quickly detected. The common PCR method of the first method was used as a control.

[0061] The kit constructed in the present invention mainly includes specific primer pair 10 μM Lm16F and 200 μL each of Lm16R, 10×PCR buffer 500 μL, 20 mM MgCl 2 Solution 300 μL, 2.0 mM dNTP 200 μL and 1 U / μL Taq-DNA polymerase 200 μL. The theoretical detection times of this kit is 200 times.

[0062] The instructions for use of the kit constructed in the present invention are as follows:

[0063] (1) Weigh 25 g of the sample under sterile conditions, add it to 225 mL of BHI medium, and cult...

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Abstract

The invention discloses a nucleotide sequence for detecting listeria monocytogenes and a detection method and a detection kit, wherein the nucleotide sequence is expressed as SEQ ID NO. 1, the detection method is as follows: firstly, extracting the gene group DNA of the sample to be detected; secondly, taking the gene group DNA as the template, mixing with the specific primer Lm16, PCR buffer solution, MgCl2 solution, deoxidation nucleoside triphosphate mixture and Taq-DNA polymerase for preparing the PCR reaction system, carrying out the PCR reaction; finally, detecting whether the PCR product is the single amplification product, the size of which is 1623bp. The kit comprises the specific primer, PCR buffer solution, MgCl2 solution, deoxidation nucleoside triphosphate mixture and Taq-DNA polymerase. The kit can effectively detect the listeria monocytogenes in the food, and the detection sensitivity for the bacteria is high, the specificity is good and the antijamming capability is strong.

Description

technical field [0001] The invention relates to the technical field of detection of food-borne pathogenic bacteria, and relates to a nucleotide sequence, a detection method and a detection method for detecting Listeria monocytogenes (Listeria monocytogenes, referred to as Listeria monocytogenes). Reagent test kit. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes, LM) is a zoonotic pathogen, belonging to the genus Listeria (Listeria spp.), widely present in nature, when livestock is infected, it passes through the food chain to the last cause infection in humans. According to WHO reports, the carrying rate of Listeria monocytogenes in feces of healthy people is 0.6% to 16%, and 70% of people can carry the bacteria for a short time. Human infection can lead to gastroenteritis, sepsis, meningitis, miscarriage and other diseases. Pregnant women, infants, the elderly and those with low immunity are susceptible groups. Humans infected with Listeria mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689
Inventor 陆兆新陶婷婷别小妹吕凤霞张充赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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