Dual PCR method for simultaneously detecting Listeria monocytogene and Listeria ivanovii

A technology for Listeria monocytogenes and Listeria yiseri, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc. Good specificity and high detection efficiency

Inactive Publication Date: 2018-05-04
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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The requirements for experimental equipment are not high, and

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  • Dual PCR method for simultaneously detecting Listeria monocytogene and Listeria ivanovii
  • Dual PCR method for simultaneously detecting Listeria monocytogene and Listeria ivanovii
  • Dual PCR method for simultaneously detecting Listeria monocytogene and Listeria ivanovii

Examples

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Embodiment 1

[0029] Example 1: Establishment of a dual real-time fluorescent quantitative PCR detection system for Listeria monocytogenes and Listeria ishii

[0030] Through the comparison of the whole genome sequences of Listeria monocytogenes and Listeria eziri, two species-specific genes were screened: LMOf2365_0970 (YP_013571.1) and AX25_00730 (AHI54712.1), and the gene LMOf2365_0970 was only found in single 100% homology within Listeria species, gene AX25_00730 is 100% homologous only to Listeria iwiri. Beacon Designer 8.0 software was used to design specific primers and probes, and blastn comparison analysis was carried out in the NCBI database, and then the PCR amplification results of the primers to be selected were simulated through the online bioinformatics tool "in silico". PCR amplification was used to verify the specificity of the primers, and the sequences of the fluorescent quantitative PCR primers were confirmed as follows:

[0031] TQM-f: 5'-cagaccgttatagtgaattc-3'. (SEQ...

Embodiment 2

[0043] Embodiment 2: Specificity evaluation of the dual real-time fluorescent quantitative PCR detection system of the present invention

[0044] Inoculate 36 strains of Listeria monocytogenes (including 13 serotypes), 1 strain of Listeria izini and 5 strains of other Listeria in the modified Listeria enrichment medium, and shake and cultivate at 37°C for 10 hours at 180rpm; Take 1 mL of the bacterial liquid in a 1.5 mL centrifuge tube, extract and purify the genomic DNA according to the conventional genome extraction method; the genomic DNA is stored at -20°C for later use. Among them, the components of the improved Listeria enrichment medium are: tryptone 17.0g / L, polyvalent peptone 3.0g / L, sodium chloride 5.0g / L, potassium dihydrogen phosphate 1.4g / L, disodium hydrogen phosphate 12.0 g / L, escin 1.0g / L, acriflavine 0.02g / L, nalidixic acid 0.018g / L, lithium chloride 0.015g / L, sodium pyruvate 0.7g / L, dissolved in 1000mL distilled water middle. ), the genomic DNA extraction a...

Embodiment 3

[0050] Example 3: Sensitivity, anti-interference and repeatability evaluation of dual real-time fluorescent quantitative PCR detection system

[0051] (1) Sensitivity evaluation

[0052] The genomic DNA of Listeria monocytogenes and Listeria eziri were serially diluted 10 times with TE buffer respectively, and the initial concentrations of the genomic DNA templates of the two bacteria were measured by an ultra-micro nucleic acid analyzer, which were 61.6ng / μL and 187.1ng / μL. 2 μL of DNA in each dilution gradient was used as a template for real-time fluorescent quantitative PCR reaction, and three parallel samples were made for each dilution gradient, and sterile water was used as a blank control. The results are shown in Table 2 and attached figure 2 As shown, when the concentration of Listeria monocytogenes is in the range of 61.6ng / μL~61.6fg / μL, the primer TQM can obtain credible positive results, and when the DNA concentration drops to 6.16fg / μL, the reaction system Th...

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Abstract

The invention discloses a dual PCR method for simultaneously detecting Listeria monocytogene and Listeria ivanovii, and belongs to the technical field of detection of foodborne pathogenic bacteria. Primers and probes are designed in allusion to a newly discovered Listeria monocytogene-specific gene LMOf2365_0970 and a newly discovered Listeria ivanovii-specific gene AX25_00730; by the detection method, the Listeria monocytogene and the Listeria ivanovii in food can be effectively detected; the detection limit for the two target bacteria is N*102 CFU/mL, and the fluorescence quantitative PCR detection time is shorter than 40 minutes; the detection method has the characteristics of high detection sensitivity, good specificity and strong anti-interference ability; by the detection method, quick qualitative and accurate quantitative detection of the Listeria monocytogene and the Listeria ivanovii in food can be achieved, and the purpose of rapidly screening contaminated food samples is achieved.

Description

technical field [0001] The invention relates to a double-PCR method for simultaneously detecting Listeria monocytogenes and Listeria iridii, belongs to the technical field of detection of food-borne pathogenic bacteria, and is a method for detecting Listeria monocytogenes and Listeria monocytogenes and Listeria monocytogenes based on molecular biological technology. Detection method of Listeria ivanovii. Background technique [0002] Within the genus Listeria, Listeria monocytogenes (LM) and Listeria ivanovii (LV) have been proven to be two typical pathogenic microorganisms that can cause Listeria in humans and animals. fungal disease, which can lead to death in severe cases. Listeria monitoring networks have been established in countries such as Europe and the United States. Every year, outbreaks of foodborne diseases caused by Listeria monocytogenes contamination in food, such as meat products, seafood, fresh milk, raw vegetables and ready-to-eat foods, are reported. Ani...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/04C12R1/01
CPCC12Q1/6851C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 陆兆新陶婷婷别小妹吕凤霞张充
Owner NANJING AGRICULTURAL UNIVERSITY
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