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Preparation and utilization method of yersinia genus rapid detection reagent kit

A technology of Yersinia and detection kit, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome operation, time-consuming, can not meet the needs of disease prevention and control, etc., and achieves high sensitivity, specific effect

Inactive Publication Date: 2008-06-18
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are three main methods for detecting Yersinia in the past: 1. Physiological and biochemical identification technology, which is time-consuming and cumbersome to operate, and cannot meet the needs of disease control; 2. Enzyme-linked immunosorbent assay, 3. Polymerase chain reaction method (PCR method), which is faster and more sensitive than the previous two methods, but requires an expensive PCR instrument
There is no kit and method for detecting Yersinia by the loop-mediated isothermal amplification method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Prepare the Loop-Mediated Isothermal Amplification Kit for Yersinia as follows:

[0037] (1) LAMP reaction solution A:

[0038] Contains 2.5 μL 10× Thermopol reaction buffer, 1.0 μL 10 mmol / L dNTP, 1.0 μL 20 μmol / L upstream internal primer (FIP), 1.0 μL 20 μmol / L downstream internal primer (BIP), 0.25 μL 20 μmol / L upstream external primer ( F3), 0.25 μL 20 μmol / L downstream outer primer (B3), 0.5 μL 100 mmol / L MgSO 4 , 12.5 μL 2mol / L betaine and 4 μL ddH 2 O (sterilized double distilled water).

[0039] The upstream internal primer 5-ACCTGTGTTCCATGCTGCTTCTATTCGCAACTGACATGTCGG-3 described therein;

[0040]Downstream internal primer 5-TAACAAAGGTCACCGTCCAGGCCAGAACGCAGGTCTTGTGAA-3;

[0041] Upstream outer primer 5-CCAGAGCCAGAGTTCTTCCT-3;

[0042] Downstream outer primer 5-GGCCCATCTCTTCCATCAC-3.

[0043] The mass ratio of the mixture of four deoxyribonucleic acids in the dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0044] The 10× Thermopol reaction buffer described therein ...

Embodiment 2

[0062] Prepare the Loop-Mediated Isothermal Amplification Kit for Yersinia as follows:

[0063] (1) LAMP reaction solution A:

[0064] Contains 2.5 μL 10× Thermopol reaction buffer, 1.0 μL 10 mmol / L dNTP, 1.0 μL 20 μmol / L upstream internal primer (FIP), 1.0 μL 20 μmol / L downstream internal primer (BIP), 0.25 μL 20 μmol / L upstream external primer (F3 ), 0.25 μL 20 μmol / L downstream outer primer (B3), 0.5 μL 100 mmol / L MgSO 4 , 12.5 μL 2mol / L betaine and 4 μL ddH 2 O (sterilized double distilled water).

[0065] Wherein the quality dUTP:dATP:dGTP:dCTP=2:1:1:1 of the mixture of four deoxyribonucleic acid in dNTP.

[0066] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0067] The 10× Thermopol reaction buffer described therein contains trihydroxymethylaminomethane-hydrochloric acid (Tris-HCl) of 200mmol / L pH8.8, 100mmol / L potassium chloride (KCl), 100mmol / L ammonium sulfate (( NH 4 )...

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Abstract

The invention relates to a preparation and using method of a yersinia genus rapid detection kit. The kit includes loop-mediated isothermal amplification reaction liquid A, BstDNA polymerase B and chromogenic reagent C. The reaction liquid A contains reaction buffer, dNTP, sulfate magnesium, upstream internal primer 5- CCGGTTTGATCGGTTTCGCCCACTTACAAGATGGGTGTGCC-3, downstream internal primer 5- GTGCGTTTCTGGCCGAGCTTGCAGACGTTTTGCCAGGATT-3, upstream external primer 5- CGCCGTGAAGGTAAAGTTCA-3, downstream external primer 5-CAGAGTTCAGGAACGACAGC-3 and betaine. The method for detecting the yersinia genus includes the DNA extraction of a sample to be detected or bacteria, the loop-mediated isothermal amplification and the chromogenic detection of the yersinia genus. The invention has the advantages of rapid property, 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 method for preparing and using a Yersinia rapid detection kit. Background technique [0002] Yersinia, also known as Pasteurella, is a genus of bacteria that includes Yersinia pestis. It can parasitize all warm-blooded animals and cause hemorrhagic sepsis. Gram-negative, aerobic or anaerobic. The fermentability of carbohydrates is very weak and does not produce gas. The genus Yersinia includes 11 species, of which three are pathogenic to humans: Yersinia enterocolitica, Pseudotuberculosis and Yersinia pestis. Only Yersinia enterocolitica and Pseudotuberculosis have been identified as foodborne pathogens. Although the bacteria of this genus have obvious specificity in terms of pathogenic hosts, the composition of antigens is very similar. It also has a very high susceptibility to bacteriopha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 陈颖高宏伟林超刘彩霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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