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Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method

A ring-mediated isothermal, cholera toxin technology, used in biochemical equipment and methods, microbial assay/test, resistance to vector-borne diseases, etc. To achieve the effect of strong technical specificity and high sensitivity

Inactive Publication Date: 2009-03-18
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing detection methods of Vibrio cholerae mainly include conventional separation and identification method, common PCR detection method, fluorescent quantitative PCR detection method, colloidal gold immunochromatography test method, etc., conventional methods are time-consuming, and colloidal gold immunochromatography test method has poor sensitivity , although PCR can make up for the above deficiencies, but its cost is higher
The loop-mediated isothermal amplification method has good specificity, high sensitivity, and can be used for rapid detection. At present, there is no report on the detection of Vibrio cholerae by the loop-mediated isothermal amplification method

Method used

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  • Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method
  • Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method
  • Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method

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Experimental program
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Effect test

Embodiment 1

[0033] Prepare the loop-mediated isothermal amplification reaction solution of cholera toxin-producing Vibrio cholerae according to the following formula:

[0034] (1) LAMP reaction solution:

[0035] Contains 2.5 μL 10× Thermopol reaction buffer, 1.4 μL 25 mmol / L dNTP (mixture of four kinds of DNA), 4.0 μL 10 μmol / L upstream internal primer (FIP), 4.0 μL 10 μmol / L downstream internal primer (BIP), 0.5 μL 10 μmol / L upstream outer primer (F3), 0.5 μL 10 μmol / L downstream outer primer (B3), 2 μL 100 mmol / L MgSO 4 , 5 μL 5M betaine and 2.1 μL ddH 2 O (sterilized double distilled water).

[0036] The upstream internal primers described therein:

[0037] 5-TGAATCCACGGCTCTTCCCT-TGGTTATGGATTGGCAGG-3,

[0038] Downstream internal primers:

[0039] 5-GGTTGTGGGAATGCTCCAAG-ACTTTGGGTTTTTTCATCGC-3,

[0040] Upstream outer primer: 5-GATATTGCTCCAGCAGCA-3,

[0041]Downstream outer primer: 5-CGTCAAGGAATTTTACACCTAG-3

[0042] The mass ratio of the four deoxyribose nucleic acids in the m...

Embodiment 2

[0057] The loop-mediated isothermal amplification kit for cholera toxin-producing Vibrio cholerae was prepared according to the following recipe:

[0058] (1) LAMP reaction solution:

[0059] Contains 2.5 μL 10× Thermopol reaction buffer, 1.4 μL 25 mmol / L dNTP, 4.0 μL 10 μmol / L upstream internal primer (FIP), 4.0 μL 10 μmol / L downstream internal primer (BIP), 0.5 μL 10 μmol / L upstream external primer (F3 ), 0.5 μL 10 μmol / L downstream outer primer (B3), 2 μL 100 mmol / L MgSO 4 , 5 μL 5mol / L betaine and 2.1 μL ddH 2 O (sterilized double distilled water).

[0060] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0061] The mass ratio of the four deoxyribose nucleic acids in the above mixture dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0062] (2) UNG enzyme: 1U / μL;

[0063] (3) Bst DNA polymerase: 8U / μL;

[0064] (4) Chromogen: 10% fluorescent dye DNAGreen.

[0065] Follow the procedures (...

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Abstract

The invention relates to a loop-mediated isothermal amplification method for rapid detection of cholera toxin vibrio cholera. A reagent comprises a loop-mediated isothermal amplification reaction liquid, Bst DNA polymerase, and a chromogenic reagent, wherein the reaction liquid contains a reaction buffer liquid, dNTP, magnesium sulfate, an upstream inner primer 5-TGAATCCACGGCTCTTCCCT-TGGTTATGGATTGGCAGG-3, a downstream inner primer 5-GGTTGTGGGAATGCTCCAAG-ACTTTGGGTTTTTTCATCGC-3, an upstream outer primer 5- GATATTGCTCCAGCAGCA-3, a downstream outer primer 5-CGTCAAGGAATTTTACACCTAG-3, and betaine. The method for detecting the cholera toxin vibrio cholera comprises the steps of the extraction of bacterial DNA, the loop-mediated isothermal amplification of the cholera toxin vibrio cholera, and chromogenic detection. The method has the advantages of rapidness, strong specificity, high sensitivity, and low cost.

Description

technical field [0001] The invention relates to a method for rapid detection of bacterial samples using a loop-mediated isothermal amplification (LAMP) technology, in particular to a rapid detection method for cholera toxin-producing Vibrio cholerae loop-mediated isothermal amplification. Background technique [0002] Vibrio cholerae (Vibrio cholerae) belongs to the Vibrio family, a Gram-negative bacterium, and the bacterium is small and curved. It is the pathogen of human cholera. Cholera is an ancient and widespread severe infectious disease. The sub-pandemic, mainly manifested as severe vomiting, diarrhea, dehydration, and a high mortality rate. Vibrio cholerae can be divided into more than 200 O serogroups (O serogroups) according to different bacterial (O) antigens, but only 01 and 0139 groups of Vibrio cholerae can cause cholera. Before 1992, only two biotypes of Vibrio cholerae (V.cholerae 01) group 01 (Classical biotype and El Tor biotype) caused seven cholera world...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 雷质文贺楠赵丽青马维兴张健姜英辉李正义房保海唐静祝素珍贾俊涛刘云国
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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