Loop-mediated isothermal amplification fast detection method of producing candida albicans fungi

A ring-mediated isothermal, Candida albicans technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of low specificity, high cost, and prone to false positives, and achieve high sensitivity, highly specific effect

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 detection methods for Candida albicans include germ tube method, NGL enzymatic method, chromogenic culture method, common PCR, fluorescent quantitative PCR and direct immunofluorescence, etc. Among them, germ tube method and NGL enzymatic method are prone to false positives, Low specificity; PCR is sensitive but has high cost
The loop-mediated isothermal amplification method can make up for these shortcomings, and there is no report on the detection of Candida albicans by the loop-mediated isothermal amplification method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Prepare the loop-mediated isothermal amplification reaction solution for Candida albicans according to the following recipe:

[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-CACATTTTGGATAAGGTTCACCATT-ATAGAAAACCTCAAGATTTCCGT-3,

[0038] Downstream internal primers:

[0039] 5-TCGTGTTCGTGAAAGTGAAGATAAT-CTTTCTATCATCCAAATCGTAGA-3,

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

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

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

Embodiment 2

[0057] Prepare the loop-mediated isothermal amplification reaction solution for Candida albicans 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 (1)-(3) below f...

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PUM

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Abstract

The invention relates to a rapid detection method for candida albicans 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-CACATTTTGGATAAGGTTCACCATT-ATAGAAACCTCAAGATTTCCGT-3, a downstream inner primer 5-TCGTGTTCGTGAAAGTGAAGATAAT-CTTTCTATCATCCAAATCGTAGA-3, an upstream outer primer 5-ACCGTTGATTTCCAATTCG-3, a downstream outer primer 5-TTAACTTGAGCCATGGAGAT-3, and glycine betaine. The method for detection of candida albicans comprises the steps of DNA extraction of the fungi, loop-mediated isothermal amplification of the candida albicans, and chromogenic detection. The method has the characteristics 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 candida albicans loop-mediated isothermal amplification. Background technique [0002] Candida albicans (Canidia Albicans) belongs to the fungal kingdom Deuteromycotina - Bacillus - Cryptococcus order - Cryptococcus family, is a biphasic single-celled yeast. In humans, the yeast cell type is often asymptomatic; the hyphae type is often present when tissue invasion and symptoms are present. Candida albicans is a parasitic bacterium that exists widely in nature and is one of the normal flora of the human body. It usually lives in the skin, mucous membranes, digestive tract and other organs of the human body. When the body's resistance decreases, Candida albicans Bacteria will multiply, and when it reaches a certain amount, the human body will get sick, so Candida albica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 雷质文贺楠姜英辉刘云国房保海李正义唐静马维兴张健祝素珍贾俊涛赵丽青
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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