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Listeria monocytogenes fluorescence quantitative PCR (Polymerase Chain Reaction) test kit and test method

A technology of mononucleosis and detection kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., and can solve the problems of inaccurate quantification of false positives, difficulties in the preparation of monoclonal antibodies, cross-reactions, etc. problems, to achieve the effects of accurate quantification, high detection specificity, and high detection sensitivity

Inactive Publication Date: 2010-08-18
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

Immunological methods are faster, but the preparation of monoclonal antibodies is difficult, prone to cross-reaction, and poor specificity
Although the conventional PCR method has the advantages of simplicity, speed, and sensitivity, it has problems such as inaccurate quantification and false positives caused by contamination caused by post-PCR post-processing. Therefore, it is urgent to develop an accurate, sensitive, fast, and pollution-free rapid detection method

Method used

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  • Listeria monocytogenes fluorescence quantitative PCR (Polymerase Chain Reaction) test kit and test method
  • Listeria monocytogenes fluorescence quantitative PCR (Polymerase Chain Reaction) test kit and test method
  • Listeria monocytogenes fluorescence quantitative PCR (Polymerase Chain Reaction) test kit and test method

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Embodiment

[0030] The components of the Listeria monocytogenes fluorescent quantitative PCR detection kit involved in this embodiment are:

[0031] (both purchased from Treasure Bioengineering (Dalian) Co., Ltd.):

[0032] Fluorescent quantitative PCR reaction solution, fluorescent probe (5μM, 1ul), hot-start TaqDNA polymerase (5U / μl, 0.2ul), standard positive template, negative quality control standard;

[0033] The fluorescent quantitative PCR reaction liquid is specifically: 10×PCR buffer 2ul, 25mM MgCl 21.5ul, 2.5mM dNTPs1.5ul, forward primer (5μM, 1ul), reverse primer (5μM, 1ul); wherein the base sequence of the forward primer is shown in SEQIDNo.1, and the base sequence of the reverse primer is shown in Shown in SEQID No.2;

[0034] The base sequence of the fluorescent probe is shown in SEQID NO: 3, wherein the fluorescent reporter group is FAM, and the fluorescent quencher group is ELIPCE;

[0035] Described standard positive template is the DNA of recombinant plasmid, and the ...

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Abstract

The invention relates to a Listeria monocytogenes fluorescence quantitative PCR (Polymerase Chain Reaction) test kit and a test method, belonging to the technical field of food safety. The test kit comprises a fluorescence quantitative PCR reaction liquid, a fluorescent probe, a warm start Taq DNA polymerase, a standard positive templet and a negative quality control standard product. The method comprises the following steps of: extracting a DNA of a sample to be tested with a common method; using the obtained DNA as a template; carrying out amplification to PCR by using the Listeria monocytogenes fluorescence quantitative PCR test kit to obtain reaction products; then placing the reaction products in a quantitative PCR instrument for fluorescence test; and carrying out a qualitative test or a quantitative test on the Listeria monocytogenes in the sample. The test kit of the invention has the advantages of high test specificity, high test sensitivity, accurate quantification and high test speed.

Description

technical field [0001] The invention relates to a kit and a detection method in the technical field of food safety, in particular to a fluorescent quantitative PCR detection kit and a detection method for Listeria monocytogenes. Background technique [0002] Listeria is a Gram-positive non-bacillus, and there are 7 species in this genus (Listeria monocytogenes, Listeria monocytogenes, Listeria sheep (L. ), Listeria (L.seeligeri), Listeria grayi (L.grayi), Listeria welschii (L.welshimeri), Listeria innocua (L.innocua)), of which Only Listeria monocytogenes is pathogenic to humans. It is a zoonotic pathogen with severe symptoms of poisoning. In addition to common gastrointestinal manifestations such as vomiting and diarrhea, it can also erode the human central nervous system ( Mainly manifested as sepsis, meningitis, miscarriage and other diseases), causing great damage to the body. Most cases of listeriosis in humans are classified as sporadic, explosive, or a combination o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04G01N21/64
CPCY02A50/30
Inventor 史贤明张丹丹龙飞王大鹏施春雷
Owner SHANGHAI JIAO TONG UNIV
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