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Design method and detection method of specific primers for resistance gene of rifampicin antibiotic resistance caused by SNP

A resistance gene and design method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/testing, can solve problems such as difficult detection and analysis, complicated operation, and long time consumption, and achieve an optimized reaction system , optimized reaction conditions, fast results

Active Publication Date: 2020-10-23
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the acquisition of long fragment gene sequences is mainly through cloning vector sequencing, but there are problems such as complicated operation and long time consumption
However, the conventional PCR reaction is difficult to amplify DNA fragments above 3kb. Even if the amplification is successful, the yield is low, and it is difficult to carry out further detection and analysis.

Method used

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  • Design method and detection method of specific primers for resistance gene of rifampicin antibiotic resistance caused by SNP
  • Design method and detection method of specific primers for resistance gene of rifampicin antibiotic resistance caused by SNP
  • Design method and detection method of specific primers for resistance gene of rifampicin antibiotic resistance caused by SNP

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Primer design

[0036] (1) The resistance gene rpoB gene contains an open reading frame of 3552bp, using the reference sequence of rpoB of Staphylococcus aureus published by NCBI (GenBank NC_007795.1:522160-525711), which encodes 1183 amino acids.

[0037] (2) Use DNAman software to design primers based on the above reference sequence to generate candidate primers.

[0038] (3) Since the full-length sequence of the rpoB gene needs to be amplified, it must be taken at the beginning and end of the reference sequence, and the upstream primers start from the beginning of the reference sequence, and the downstream primers end from the end of the reference sequence. Therefore, some primers are designed The software cannot automatically generate specific primers. It is necessary to use the software and combine some long-range primer design principles to screen the primers. The design of long fragment primers must pay attention to the following issues:

[0039] a. Primer length: 25...

Embodiment 2

[0051] Specific primer pair amplification experiment

[0052] (1) Extract the genomic DNA of 6 strains of Rifampicin with different minimum inhibitory concentration (MIC) values ​​of Staphylococcus aureus (denoted as Li 0~Li 5), 1 sample of activated sludge and 1 strain of E. coli ATCC25922.

[0053] (2) Using each genomic DNA extracted in step 1 as a template, perform PCR amplification with the specific primer pair screened in Example 1 to obtain the corresponding PCR product; perform agarose gel electrophoresis on the PCR product;

[0054] Among them, the PCR reaction system is:

[0055] The total reaction system is 50μL

[0056]

[0057] The PCR amplification program is: 94°C pre-denaturation 5min; 94°C, 2min, 97°C, 1min, 60°C, 1min, 35 cycles; 68°C, 5min, 68°C, 10min.

[0058] (3) Recover the PCR products and send them to BGI for sequencing.

[0059] Reference for agarose gel electrophoresis results figure 1 . Lanes 1 to 6 represent 6 strains of Staphylococcus aureus, lane 7 represen...

Embodiment 3

[0067] This embodiment provides a method for screening rifampicin-resistant strains, which includes the following steps:

[0068] (1) Obtain the genomic DNA of the strain to be screened;

[0069] (2) Use the specific primer pairs, DNA polymerase, nucleotides, etc. in Example 1 to extend the genomic DNA according to the amplification procedure in Example 2;

[0070] (3) Sequencing the amplified product after recovery, comparing it with the rpoB gene sequence of wild-type Staphylococcus aureus, obtaining the SNP locus information of the rpoB gene of the strain to be screened, and screening rifampicin based on its SNP locus information Resistant strains.

[0071] The specific primer pair used in this solution can accurately amplify the full-length sequence of the rpoB gene through conventional PCR, so that the strain screening can be effectively achieved.

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Abstract

The invention provides a design method and a detection method of specific primers for a resistance gene of rifampicin antibiotic resistance caused by SNP. The design method comprises the following steps: step 1, acquiring a nucleotide sequence of the resistance gene; step 2, performing primer design on the nucleotide sequence to generate candidate primers; and step 3, performing screening according to the parameters of the candidate primers to obtain a specific primer pair. The design method provided by the embodiment of the invention at least has the following beneficial effects: by setting and screening the primer parameters, the designed specific primer pair has higher specificity, and the designed specific primer pair can directly obtain the full-length sequence of the resistance genethrough PCR amplification, thereby greatly simplifying the operation steps.

Description

Technical field [0001] The present invention relates to the technical field of biological detection, in particular to a method for designing and detecting specific primers for rifampicin antibiotic resistance genes caused by SNP. Background technique [0002] Rifampicin is a rifamycin semi-synthetic broad-spectrum antibacterial drug. It has antibacterial activity against various pathogenic microorganisms such as Staphylococcus aureus and Mycobacterium tuberculosis. It is mainly used in the treatment of tuberculosis and other tuberculosis. In recent years, due to the rising incidence of tuberculosis and the abuse of rifampicin, the content of rifampicin in the environment has increased year by year, which has also led to a significant increase in the detection rate of rifampicin-resistant strains in the environment. [0003] Rifampicin specifically binds to bacterial DNA-dependent RNA polymerase subunits, inhibits the activity of RNA polymerase, interferes with the transcription ini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q2600/156Y02A50/30
Inventor 李炳徐结梁贺彬张家禹雷华新
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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