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LAMP detection kit and application

A detection kit and the technology of the kit, which are applied in the biological field, can solve the problems of reducing reaction amplification efficiency, consumption of reaction substrates, primer mismatches, etc., and achieve the effects of improving amplification efficiency, improving sensitivity, and preventing base stacking.

Inactive Publication Date: 2017-04-26
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem that there are too many LAMP primers and primer mismatches are prone to occur, the reaction substrate is consumed in the reaction, the amplification efficiency of the reaction is reduced, and even false positives appear in the labeling results of the test strips. The present invention provides a LAMP The detection kit adopts the following technical scheme:

Method used

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  • LAMP detection kit and application
  • LAMP detection kit and application

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Experimental program
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Effect test

Embodiment 1

[0071] Embodiment 1: Optimization of LAMP reaction system

[0072] Strain cultivation and DNA extraction:

[0073] Kronobacter sakazakii ATCC29544 was selected as the experimental strain for related experiments. The strain was taken out from the refrigerator stored at -80°C, inoculated into TSB liquid medium at an inoculation amount of 3%, activated for 12 hours, and then TSA solid medium Three-section line was drawn on it, cultured for 16 hours, a single colony was picked in TSB liquid medium, and DNA was extracted after 12 hours.

[0074] Use the water bath method to extract the DNA of the cultured bacterial liquid, centrifuge at 10,000r / min for 3min, add 50uL of TE buffer, bathe in water at 100°C, and put it in ice bath for 5min after 10min, then centrifuge at 10,000r / min, take the supernatant for 1min, and the supernatant is ready for the DNA template.

[0075] 1. Addition effect and dosage optimization of Taq SSB protein

[0076] LAMP amplification reaction system: Eac...

Embodiment 2

[0096] Embodiment 2: the preparation of kit

[0097] The object of the present invention is to provide a kind of LAMP detection kit, and this kit comprises LAMP amplification reaction system and colloidal gold test strip; LAMP amplification reaction system comprises LAMP primer set, LAMP amplification reaction solution; LAMP primer set includes Primers FIP and BIP, outer primers F3 and B3, loop primers LF and LB; LAMP amplification reaction solution contains Taq SSB protein, Bst DNA polymerase, Bst buffer, dNTPs, Mg 2+ , template and ddH 2 O.

[0098] The components of each 25uL LAMP amplification reaction system are as follows: inner primers FIP and BIP, outer primers F3 and B3, loop primers LF and LB, 3ng / μL Taq SSB protein, 0.6μL 8U Bst DNA polymerase, 1μL 10×Bst buffer, 0.8mM-2.8mM dNTPs, 1mM-5mM Mg 2+ , 1 μL DNA template; the concentration ratio of the outer primer and the inner primer is 1:3; 2 O make up.

[0099] Any two primers in the LAMP primer set are directly ...

Embodiment 3

[0104] Embodiment 3: the preparation of kit

[0105] The object of the present invention is to provide a kind of LAMP detection kit, and this kit comprises LAMP amplification reaction system and colloidal gold test strip; LAMP amplification reaction system comprises LAMP primer set, LAMP amplification reaction solution; LAMP primer set includes Primers FIP and BIP, outer primers F3 and B3, loop primers LF and LB; LAMP amplification reaction solution contains Taq SSB protein, Bst DNA polymerase, Bst buffer, dNTPs, Mg 2+ , template and ddH 2 O.

[0106] The components of each 25 μL LAMP amplification reaction system are as follows: inner primers FIP and BIP, outer primers F3 and B3, loop primers LF and LB, 5 ng / μL Taq SSB protein, 1 μL 8U Bst DNA polymerase, 3 μL 10×Bst buffer, 2.8mM dNTPs, 5mM Mg 2+ , 2 μL DNA template; the ratio of the concentration of the outer primer to the inner primer is 1:8; the ratio of the concentration of the outer primer to the loop primer is 1:5, ...

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Abstract

The invention discloses a LAMP detection kit and application, and belongs to the technical field of biology. The kit provided by the invention comprises a LAMP amplification reaction system and a colloidal gold test strip, wherein the LAMP amplification reaction system comprises a LAMP primer group and LAMP amplification reaction liquid; the LAMP primer group comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; the LAMP amplification reaction liquid contains Taq SSB proteins, Bst DNA polymerase, Bst buffer, dNTPs, Mg<2+>, a template and ddH2O. The kit has the advantages of easiness in operation, low detection cost, short detection time, high specificity and high sensitivity; by adopting the kit, the primers can be prevented from generating polymers, the generation of primer dimers is reduced or prevented, the generation of false positive is prevented, and meanwhile the amplification efficiency of a LAMP reaction is increased.

Description

technical field [0001] The invention relates to a LAMP detection kit and its application, belonging to the field of biotechnology. Background technique [0002] Cronobacter, after re-identification and typing in 2008, can be divided into: Cronobacter akazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter condimenti, Cronobacter universalis, Cronobacter dublinensis. Although the pathogenic events caused by Cronobacter are rare, once it occurs, it will lead to a very high morbidity rate. The pathogenicity caused by Cronobacter can cause severe neonatal meningitis, small intestine and colon Diseases such as inflammation and sepsis pose a great threat to infant milk powder. Although not all species can cause diseases in infants and young children, the 2016 International Microbiological Standards recommends that all Cronobacter species should not be detected in infant milk powder, so it is particularly important to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6804C12Q1/689C12Q2531/119C12Q2565/625C12Q2522/101C12Q2563/131
Inventor 姜毓君姜霞潘瑞丽满朝新李明雨赵玥明曲艳艳
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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