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Fast detecting reagent kit for enterobacter sakazakii and detecting method thereof

A technology of Enterobacter sakazakii and detection kit, which is applied in the rapid detection kit of Enterobacter sakazakii and its detection field, achieving high sensitivity and strong specificity

Inactive Publication Date: 2008-12-10
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

There is no report on the detection kit and detection method of Enterobacter sakazakii by 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 kit of Enterobacter sakazakii was made 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 100mmol / L MgSO 4 , 5μL 5M Betaine and 7.6μL ddH 2 O.

[0036] The upstream inner primer:

[0037] 5-TATGCGGGATCGAACCGCAGA-GGCTATAGCTCAGCTGGGA-3,

[0038] Downstream inner primer:

[0039] 5-GCTCCACCATCACTTCGGAGTG-TTCAGCTTGTTCCGGATTGT-3,

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

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

[0042] The mass ratio of the four kinds of deoxyribonucleic acids in the mixture dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0043] (2) UNG enzyme: 1U / μL;

[0044...

Embodiment 2

[0057] The loop-mediated isothermal amplification kit of Enterobacter sakazakii was made according to the following formula:

[0058] (1) LAMP reaction solution:

[0059] Contains 2.5μL 10×Thermopol reaction buffer, 1.0μL 10mmol / L dNTP, 1.0μL 20μmol / L upstream internal primer (FIP), 1.0μL 20μmol / L downstream internal primer (BIP), 0.25μL 20μmol / L upstream external primer (F3 ), 0.25μL 20μmol / L downstream external primer (B3), 0.5μL 100mmol / L MgSO 4 , 12.5μL 2mol / L betaine and 4μL 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 above-mentioned mixture dNTP is the same as above.

[0062] (2) UNG enzyme: 1U / μL;

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

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

[0065] Perform the test according to the following (1)-(3) procedures:

[...

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Abstract

The invention relates to a rapid detection reagent kit for Enterobacter sakazaii by using a loop-mediated isothermal amplification technology and a detection method thereof. A loop-mediated isothermal amplification reaction liquid, Bst DNA polymerase and a chromogenic reagent are placed in the reagent kit; and the reaction liquid contains a reaction buffer liquid, dNTP, magnesium sulfate, an upstream inner primer 5- TATGCGGGATCGAACCGCAGA-GGCTATAGCTCAGCTGGGA-3, a downstream inner primer 5- GCTCCACCATCACTTCGGAGTG-TTCAGCTTGTTCCGGATTGT-3, an upstream outer primer 5- TCCGCAGGAGTTGAAGAGG-3, a downstream outer primer 5-CAGCAGCGTGTCTGTTTCA-3 and lycine. The method for detecting the Enterobacter sakazaii comprises the extraction of bacterial DNA, the loop-mediated isothermal amplification of the Enterobacter sakazaii, and chromogenic detection. The rapid detection reagent kit and the detection method have the advantages of quickness, 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 rapid detection kit for Enterobacter sakazakii and a detection method thereof. Background technique [0002] Enterobacter sakazaii (Enterobacter sakazaii) is a gram-negative abacillus parasitic in the intestines of humans and animals. As a species of Enterobacteriaceae, it has been called Enterobacter cloacae until 1980. Enterobacter. This bacteria is one of the normal intestinal flora, which can cause disease in humans and animals under certain conditions, so it is called "conditional pathogenic bacteria". Enterobacter sakazakii has a wide range of natural sources, and can exist in water, soil, plant roots, animal intestines and even processed foods. Among them, infant formula is the main channel for infants to be infected with Enterobacter sakazakii. This kind of bacteria is the most harmful to prema...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/10C12Q1/68
CPCY02A50/30
Inventor 雷质文陈颖高宏伟贺楠房保海贾俊涛姜英辉赵丽青刘云国
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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