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 the effect of strong specificity and high sensitivity

Inactive Publication Date: 2011-05-11
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 for Enterobacter sakazakii was prepared 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 (a mixture of four kinds of DNA), 2.0 μL 10 μmol / L upstream internal primer (FIP), 2.0 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), 1.5 μL 100 mmol / L MgSO 4 , 5 μL 5M betaine and 7.6 μL ddH 2 O.

[0036] The upstream internal primers described therein:

[0037] 5-TATGCGGGATCGAACCGCAGA-GGCTATAGCTCAGCTGGGA-3,

[0038] Downstream internal primers:

[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 deoxyribose nucleic acids in the mixture dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0043] (...

Embodiment 2

[0057] The loop-mediated isothermal amplification kit for Enterobacter sakazakii was prepared according to the following recipe:

[0058] (1) LAMP reaction solution:

[0059] 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).

[0060] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0061] The mass ratios of the four deoxyribonucleic acids in the mixture dNTP are the same as above.

[0062] (2) UNG enzyme: 1U / μL;

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

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

[0065] Follow the procedures (1)-(3) below for testing: ...

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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 a 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 non-bacillus bacterium parasitic in the intestines of humans and animals. Enterobacter sakii. This bacterium is one of the normal intestinal bacteria, which can cause human and animal diseases 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. Infant formula milk powder is the main channel for infants to be infected with Enterobacter sakazakii. This bacterium is most harmful to premature infants, infants with low birth weight, or infants with immunocomp...

Claims

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Application Information

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Patent Type & Authority Patents(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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