Primer pair for detecting listeria monocytogenes and method for detecting listeria monocytogenes

A technology for mononucleosis and Listeria, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the time-consuming and labor-intensive preparation of the biochemical characteristics of the medium, and is not suitable for the needs of rapid food detection, etc. problems, to achieve the effects of short detection time, simple result judgment and specific detection results

Inactive Publication Date: 2015-02-04
SHANGHAI UNIV
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  • Description
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  • Application Information

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Problems solved by technology

At present, the traditional plate culture method is still used for the detection of Listeria monocytogenes in food in my country. However, during the detection process, it is time-consuming and labor-intensive to prepare the medium, count the number of colonies, and identify the biochemical characteristics of the colonies, and the success of the culture method depends on Due to the number and status of bacteria in the sample, the sensitivity of the medium, the incubation conditions and the selectivity of the separation medium, it is not suitable for the current rapid detection of food

Method used

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  • Primer pair for detecting listeria monocytogenes and method for detecting listeria monocytogenes
  • Primer pair for detecting listeria monocytogenes and method for detecting listeria monocytogenes

Examples

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Embodiment 1

[0046] Establishment of PCR detection method for Listeria monocytogenes

[0047] Step 1, design primers according to the conserved sequence in the genomic DNA sequence of Listeria monocytogenes, and find the specific gene lmo0202 from the genome sequence of Listeria monocytogenes hly The gene is used as the target gene of the Listeria monocytogenes gene, and the gene sequence is shown in hlyID NO:1;

[0048] Enter this gene sequence into the primer design software Primer Premier 5.0 to design primers, and the product size ranges from 100 to 500 bp, and then screen for suitable primers.

[0049] hly-F:5'-ATC AAC CAg ATg TTC TCC CTg TA-3'(hly ID NO:2)

[0050] hly-R:5'- gAT TCA CTg TAA gCC ​​ATT TCg TC-3'(hly ID NO:3)

[0051] Step 2, Preparation of DNA template

[0052] 39 strains of Listeria monocytogenes and 8 negative strains (Listeria ovis, Listeria innocuci, Listeria seir, Listeria gasseri, Listeria weideri, Salmonella choleraesuis , Salmonella typhimurium, Serratia mar...

Embodiment 2

[0060] Example 2: Accuracy evaluation of PCR detection method for Listeria monocytogenes

[0061] Using the PCR detection method for Listeria monocytogenes established in Example 1, detect whether artificially contaminated meat samples contain Listeria monocytogenes. Pick a single colony of Listeria monocytogenes from the LB plate, insert it into 50mL LB liquid medium, and incubate at 37°C for 12h. Make a 10-fold gradient dilution with normal saline, and take 1 mL of dilution to 10 -8 The bacterial liquid was counted on the plate, and the initial concentration of pure culture of Listeria monocytogenes was calculated. 40 pork samples, aseptic sampling 25mL, insert 1mL 10 -2 Serially diluted Listeria monocytogenes suspension. Add 40 artificially contaminated samples to 225 mL LB liquid medium. Bacteria were enriched in a shaker (120r / min) at 37°C. Sampling was performed once after 12 hours. Take 1 mL of each sample, put it into a 1.5 mL centrifuge tube, and centrifuge a...

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Abstract

The invention relates to a primer pair for detecting listeria monocytogenes and a method for detecting the listeria monocytogenes. An upstream primer in the primer pair is a base sequence represented by SEQ ID NO: 2; and a downstream primer is a base sequence represented by SEQ ID NO: 3. When the method is used for detecting the listeria monocytogenes, the detection time is short and the cost is low; and a detection target has very strong specificity, specific detection result and simple result judgment.

Description

technical field [0001] The invention relates to a primer pair for detecting Listeria monocytogenes and a detection method thereof. Background technique [0002] Listeria monocytogenes ( Listeria monocytogenes ) referred to as Listeria monocytogenes is an important food-borne zoonotic pathogenic bacteria. It is widely distributed in nature and exists in meat, poultry, seafood, dairy products and vegetables. It can cause listeriosis in humans and animals. After infection, it mainly manifests as sepsis, meningitis and mononucleosis, especially for pregnant women, newborns, the elderly and immunodeficiency patients. Infected people can also experience flu-like symptoms, but this is less common. After pregnant women are infected, if they are not treated properly, it will cause bacteremia and lead to serious consequences such as premature delivery and dead babies. Although Listeria tenzing rarely occurs in food poisoning and infection incidents, its fatality rate is relatively...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 刘战民朱佳超
Owner SHANGHAI UNIV
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