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A primer for the detection of Bacillus subtilis by loop-mediated isothermal amplification and its application

A Bacillus subtilis, ring-mediated isothermal technology, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of less development and application reports, achieve short detection cycle, high sensitivity, and results reliable effect

Active Publication Date: 2020-05-12
SHANDONG INST FOR PROD QUALITY INSPECTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the development and application of Bacillus subtilis-related LAMP technology

Method used

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  • A primer for the detection of Bacillus subtilis by loop-mediated isothermal amplification and its application
  • A primer for the detection of Bacillus subtilis by loop-mediated isothermal amplification and its application
  • A primer for the detection of Bacillus subtilis by loop-mediated isothermal amplification and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Extract the genomic DNA of the sample to be tested, use the extracted DNA as a template, add outer primer F3 / B3, inner primer FIP / BIP and loop primer LF / LB for LAMP amplification, the total volume of the LAMP detection reaction system is 20 μL, including 0.2 μM outer primer F3 / B3, 1.6 μM inner primer FIP / BIP and 0.4 μM loop primer LF / LB, LAMP reaction mixture 10 μL, DNA template 2.0 μL, make up to 20 μL with sterilized ultrapure water. React at 65°C for 35 minutes, and then detect the reaction result.

Embodiment 2

[0049] Extract the genomic DNA of the sample to be tested, use the extracted DNA as a template, add outer primer F3 / B3, inner primer FIP / BIP and loop primer LF / LB for LAMP amplification, the total volume of the LAMP detection reaction system is 20 μL, including 0.2 μM Outer primer F3 / B3, 1.6 μM inner primer FIP / BIP and 0.4 μM loop primer LF / LB, LAMP reaction mixture 10 μL, DNA template 2.0 μL, make up to 20 μL with sterilized ultrapure water. React at 61°C for 30 minutes, and then detect the reaction result.

Embodiment 3

[0051] Extract the genomic DNA of the sample to be tested, use the extracted DNA as a template, add outer primer F3 / B3, inner primer FIP / BIP and loop primer LF / LB for LAMP amplification, the total volume of the LAMP detection reaction system is 20 μL, including 0.2 μM outer primer F3 / B3, 1.6 μM inner primer FIP / BIP and 0.4 μM loop primer LF / LB, LAMP reaction mixture 10 μL, DNA template 2.0 μL, make up to 20 μL with sterilized ultrapure water. React at 63°C for 60 minutes, and then detect the reaction result.

[0052] The primers of Bacillus subtilis detected by the loop-mediated isothermal amplification technique in the above-mentioned embodiments 1-3, i.e. the nucleotide sequences of the outer primer F3 / B3, the inner primer FIP / BIP and the loop primer LF / LB are as follows:

[0053] F3: 5'-GCGATTATTCCAGTCACGG-3';

[0054] B3: 5'-CGTTTTTCGTTCCAATGATGA-3';

[0055] FIP: 5'-CGAATTGAGATAGCGATGTTCGTTTATGCGGAGCTTTACCCTCT-3';

[0056] BIP: 5'-ATATCCGCAACAATGGTCTAATTGCTGTTTTGTGCCGT...

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Abstract

The invention discloses primers for detecting bacillus subtilis through a loop-mediated isothermal amplification method. The primers are designed and obtained with the gyrA gene and gyrB gene of the bacillus subtilis as target sequences and include a group of external primers F3 / B3, a group of internal primers FIP / BIP and a group of loop primers LF / LB. The invention further discloses application of the primers to detecting the bacillus subtilis. It is proved through experiments that the three groups of LAMP primers have quite high specificity and has the sensitivity at least one magnitude order higher than that of an existing method in a contrast test. The detecting process only needs to be conducted under the constant-temperature water bath conditions without the aid of expensive variable-temperature instruments, the result can be judged with naked eyes or by adding a simple indication agent, the reaction time is 40 minutes or shorter, the detection cycle is shorter, and the experiment cost is lower.

Description

technical field [0001] The invention belongs to the technical field of detection or identification of Bacillus subtilis in microbial fertilizers, and relates to a primer for rapid detection of Bacillus subtilis using loop-mediated isothermal amplification (LAMP) technology and an application thereof. Background technique [0002] At present, the detection methods for bacteria mainly include bacterial isolation and culture identification methods, immunological detection methods and molecular biology methods. [0003] The identification method of bacteria isolation and culture is to use liquid medium, semi-solid medium or solid medium to cultivate the isolated bacteria, and to diagnose the microorganisms through physiological, biochemical and staining microscopy. The disadvantage of this identification method is that the test cycle is very long. Generally, it takes 2 days to isolate and cultivate microorganisms, and the time required for identification is about one week. Ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/125
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 张娟杨春玉吕佩文程彬
Owner SHANDONG INST FOR PROD QUALITY INSPECTION
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