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Molecular marker for rapidly detecting listeria monocytogenes

A technology of Listeria monocytogenes and molecular markers, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve problems such as complicated operation, inability to adapt to rapid detection, and long time consumption

Active Publication Date: 2015-06-03
乐普(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a long time, the detection method of Listeria monocytogenes has always been the traditional culture method. In this method, the bacteria are first enriched, and the suspicious colonies obtained by isolation and culture are used for biochemical reaction experiments, hemolysis experiments, and cooperative hemolysis experiments (CAMP) and other immunological tests. , this method needs 7 to 11 days to isolate and identify Listeria, the operation is complex, time-consuming, and cannot meet the needs of actual inspection work and the requirements of rapid detection

Method used

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  • Molecular marker for rapidly detecting listeria monocytogenes
  • Molecular marker for rapidly detecting listeria monocytogenes
  • Molecular marker for rapidly detecting listeria monocytogenes

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, utilize PCR method to detect Listeria monocytogenes,

[0044]The primers were RliA(F) and RliA(R), RliB(F) and RliB(R), RliC(F) and RliC(R), RliE(F) and RliE(R), RliF(F) and RliF( R), ssrA(F) and ssrA(R), LhrA(F) and LhrA(R) seven pairs.

[0045] The sequences of the remaining 6 pairs of primers are:

[0046] RliA(F):gCgATTCCAgTCATTTTTATAAAg

[0047] RliA(R):gATgAAATgTCCgATTCTTgTCATg

[0048] RliC(F):gCATAATgCTTAAgTggggTAAg

[0049] RliC(R):gCgACAACCAATTTACTTgATTAC

[0050] RliE(F):gCAACAAAAACACCTATTTTCATTAgTgg

[0051] RliE(R):gCAAATAATCTTTACgAAACggAAAC

[0052] RliF(F):CgATAAACAAATTCTCCTTAAATAAAAAC

[0053] RliF(R):CAggAACAACCgATAgTAgCggg

[0054] ssrA(F):gCTTTCAAAgAAAACAgCgATTATCCg

[0055] ssrA(R):gCACTTAACAggCAAAgAAATTTTg

[0056] LhrA(F):gCTTATTAAATTATTCCgCCTATAAAg

[0057] LhrA(R): ATTTTTACTAATgTgATTTTCATgggg.

[0058] Templates: L.monocytogenes, L.innocua, L.welshimeri, L.seeligeri, Listeria grayi (L. grayi), Listeria ivanovii (L. ivan...

Embodiment 2

[0067] Example 2, verify the feasibility of bacterial liquid instead of extracting DNA for verification

[0068] Using RliB(F) and RliB(R) of the present invention as primers; ), Listeria welshi (L.welshimeri), Listeria sierra (L.seeligeri), Listeria grayi (L.grayi) bacterial solution (concentration of 50ng / μL) was directly added as a template In the PCR system (PCR reaction system and PCR reaction procedure are the same as in Example 1), PCR is performed, and 10 μL is taken for agarose gel electrophoresis.

[0069] The result is as image 3 shown; according to image 3 , we learned that: the present invention can directly use the bacterial solution instead of extracting the genome to carry out PCR verification.

Embodiment 3

[0070] Embodiment 3, template is changed into: monocytogenes Listeria (L.monocytogenes), Escherichia coli, Bacillus subtilis and the bacterium liquid of Agrobacterium (concentration is 50ng / μL), with RliB of the present invention (F ) and RliB (R) as primers; the rest are the same as in Example 1.

[0071] The result is as figure 2 mentioned.

[0072] according to figure 2 , we learned that the molecular marker designed in the present invention has certain specificity, and the band can only be obtained under the premise of using Listeria monocytogenes as a template.

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Abstract

The invention discloses a molecular marker for rapidly detecting the listeria monocytogenes. The primers of the molecular marker are as follows: RliB(F)gAAATAgCTCTTTgAggTAAgATggg; RliB(R)gCATAATCCTATgTATAACAggTTTTC. The invention further discloses a method for rapidly detecting the listeria monocytogenes by using the molecular marker. The method comprises the following steps: performing PCR amplification by taking the bacterium liquid to be tested as a template and performing agarose gel electrophoresis on the obtained PCR amplified product; when the electrophoresis result shows that a specific strip is generated in about 500 bp, judging whether the sample to be detected contains the listeria monocytogenes; and otherwise, the sample does not contain the listeria monocytogenes.

Description

technical field [0001] The invention relates to a molecular marker for rapid detection of Listeria monocytogenes and its application. Background technique [0002] In my country, the incidence of bacterial food poisoning among foodborne diseases takes the first place. According to statistics, the annual bacterial food poisoning incidents in my country account for 30% to 90% of the total number of food poisoning incidents, and the number of people accounts for 60% to 60% of the total number of food poisoning incidents. 90%, bacterial food poisoning seriously endangers people's health. [0003] Listeria monocytogenes (L.monocytogenes, Listeria monocytogenes) is a pathogenic bacterium that can cause zoonotic diseases, especially serious harm and death to pregnant women, newborns, the elderly and immunocompromised patients High rate. At present, there are 6 species of Listeria recognized internationally, namely Listeria monocytogenes (L.monocytogenes), Listeria ivanovii (L.ivan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689
Inventor 聂作明吴程程王丹叶小慧盛清
Owner 乐普(北京)生物科技有限公司
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