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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
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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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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:
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Abstract
The invention relates to a loop-mediated isothermal amplification method for rapid detection of choleratoxinvibriocholera. A reagent comprises a loop-mediated isothermal amplification reaction liquid, Bst DNApolymerase, and a chromogenicreagent, wherein the reaction liquid contains a reaction buffer liquid, dNTP, magnesiumsulfate, 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 choleratoxinvibrio cholera comprises the steps of the extraction of bacterial DNA, the loop-mediated isothermal amplification of the cholera toxinvibrio 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...
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