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Loop-mediated isothermal amplification fast detection method of producing ariatoxin fungi

A ring-mediated isothermal and aflatoxin technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of complex procedures, long cycle, high cost, etc., and achieve high sensitivity and specificity

Inactive Publication Date: 2009-03-11
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing aflatoxin detection methods are many, such as thin-layer chromatography, liquid chromatography, radioimmunoassay, enzyme-linked immunoassay, immunochromatography, common PCR, fluorescent quantitative PCR, etc., but these methods also have Disadvantages, such as long cycle, complicated procedure, high cost
The loop-mediated isothermal amplification method can better make up for these deficiencies, and there is no report on the detection of aflatoxin-producing fungi by the loop-mediated isothermal amplification method.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The loop-mediated isothermal amplification reaction solution of aflatoxin-producing fungi was prepared according to the following formula:

[0034] (1) LAMP reaction solution:

[0035] Contains 2.5μL 10×Thermopol reaction buffer, 1.4μL 25mmol / L dNTP (a mixture of four kinds of deoxyribonucleic acid), 2.0μL 10μmol / L upstream internal primer (FIP), 2.0μL 10μmol / L downstream internal primer (BIP), 0.5μL 10μmol / L upstream external primer (F3), 0.5μL 10μmol / L downstream external primer (B3), 1.5μL 100mol / L MgSO 4 , 5μL 5M Betaine and 6.6μL ddH 2 O (sterilized double distilled water).

[0036] The upstream inner primer:

[0037] 5-CGACAGTTGTTGGGGCCTGC-TCCAGGACATGTGCAGACT-3,

[0038] Downstream inner primer:

[0039] 5-CAGACAGTGTGGCAGGCATCT-CCGTGTGGATAACGAAGTGC-3,

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

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

[0042] The mass ratio of the four kinds of deoxyribonucleic acids in the mixture dNTP is dUTP:dATP:dGTP...

Embodiment 2

[0057] Prepare the loop-mediated isothermal amplification reaction solution of Enterobacter sakazakii according to the following formula:

[0058] (1) LAMP reaction solution:

[0059] Contains 2.5μL 10×Thermopol reaction buffer, 1.4μL 25mmol / L dNTP, 2.0μL 10μmol / L upstream internal primer (FIP), 2.0μL 10μmol / L downstream internal primer (BIP), 0.5μL 10μmol / L upstream external primer (F3 ), 0.5μL 10μmol / L downstream external primer (B3), 1.5μL 100mmol / L MgSO 4 , 5μL 5mol / L betaine and 6.6μL ddH 2 O (sterilized double distilled water).

[0060] The upstream inner primer, downstream inner primer, upstream outer primer, and downstream outer primer are the same as above.

[0061] The mass ratio of the four kinds of deoxyribonucleic acids in the 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) Color developer: 10% fluorescent dye DNAGreen.

[0065] Perform the test according to the following (1)-(3) procedu...

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Abstract

The invention relates to a rapid detection method for aflatoxin-producing fungi by loop-mediated isothermal amplification. Reagents used by the method comprises a loop-mediated isothermal amplification reaction solution, a Bst DNA polymerase and a color development reagent, wherein the reaction solution contains reaction buffer, dNTP, magnesium sulfate, an upstream inner primer 5-CGACAGTTGTTGGGGCCTGC-TCCAGGACATGTGCAGACT-3, a downstream inner primer 5-CAGACAGTGTGGCAGGCATCT-CCGTGTGGATAACGAAGTGC-3, an upstream outer primer 5-TCACGGCCTTCATCATCGA-3, a downstream outer primer 5-ACCAGGGGAGTTGAGATCC-3, and glycine betaine. The method for detection of aflatoxin-producing fungi comprises the steps of DNA extraction of the fungi, loop-mediated isothermal amplification of the aflatoxin-producing fungi, 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 loop-mediated isothermal amplification (LAMP) technology, in particular to a method for rapid detection of aflatoxin-producing fungus loop-mediated isothermal amplification. Background technique [0002] Aflatoxins are a class of compounds with similar chemical structures, all of which are derivatives of dihydrofuranocoumarin. In 1993, aflatoxins were classified as Class 1 carcinogens by the Cancer Research Institute of the World Health Organization (WHO) , Is a highly toxic and highly toxic substance. Aflatoxins are mainly secondary metabolites produced by Aspergillusflavus and A.parasiticus. Aflatoxins are most likely to appear in food and feed in hot and humid regions. Aflatoxins can produce fluorescence under ultraviolet light. According to the different fluorescence colors, they can be divided into two categories: B group and G group and their derivatives. Among them, B1 i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 雷质文贺楠李正义祝素珍房保海唐静贾俊涛赵丽青马维兴张健姜英辉刘云国
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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