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LAMP detection primer, detection kit and detection method for 16S rRNA methylase rmtB

A technology for detecting kits and methylases, applied in DNA/RNA fragments, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of complex operation, long detection time, and high detection cost, and achieve simple operation methods , high sensitivity and strong specificity

Active Publication Date: 2015-07-22
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of PCR amplification prone to false positives, high detection cost, complicated operation, and long detection time, and to fill the blank of using LAMP to detect the aminoglycoside antibiotic resistance gene 16S rRNA methylase rmtB, the present invention provides 16S rRNA LAMP detection primers, detection kit and detection method for methylase rmtB

Method used

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  • LAMP detection primer, detection kit and detection method for 16S rRNA methylase rmtB
  • LAMP detection primer, detection kit and detection method for 16S rRNA methylase rmtB
  • LAMP detection primer, detection kit and detection method for 16S rRNA methylase rmtB

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of a kit for detecting the aminoglycoside antibiotic resistance gene 16S rRNA methylase rmtB

[0034] (1) Design of detection primers

[0035] According to the conserved sequence of the rmtB gene known in NCBI, a pair of specific inner primers FIP and BIP, a pair of specific outer primers F3 and B3, and a loop primer L were designed using Primer 5.0 software to target aminoglycoside antibiotics. LAMP detection of drug resistance gene 16S rRNA methylase rmtB:

[0036] Outer primer F3: 5'-CGATGCCCTCACCTCCAT-3' (SEQ No.1);

[0037] Outer primer B3: 5'-GCCTTTTTTACATCGCCCG-3' (SEQ No.2);

[0038] Internal primer FIP: 5'-ATTCCTCAGTCAGGATGCGCCGGCCTCAAAAAAATACCGCG-3' (SEQ No.3);

[0039] Internal primer BIP: 5'-CCAAACAGACCGTAGAGGCTGCCAGCCTTGAGCGATTCCG-3' (SEQ No.4);

[0040] Loop primer L: 5'-GCTGCATGGAATTTGCGGGG-3' (SEQ No. 5).

[0041] (2) Detection Kit

[0042] A kit for detecting the aminoglycoside antibiotic resistance gene 16S rRNA methylase r...

Embodiment 2

[0049] Example 2: Detection method of aminoglycoside antibiotic resistance gene 16S rRNA methylase rmtB by loop-mediated isothermal amplification method

[0050] (1) Extract the genomic DNA of the clinical strain to be tested

[0051] After culturing the clinical strain to be tested overnight, take 1mL of the bacterial solution and centrifuge at 12000rpm for 2min, discard the supernatant thoroughly, take the precipitate and add 1mL sterile double distilled water, bathe in boiling water for 10min, centrifuge at 12000rpm for 2min, take the supernatant into a new centrifuge tube , measure and adjust the DNA concentration to 500ng / μL, and store at -20°C for later use.

[0052] (2) LAMP amplification

[0053] Using the kit in Example 1, set up a detection tube, a positive control tube and a negative control tube. Add 2 μL of the DNA sample to be tested into the detection tube, add 2 μL of Escherichia coli genomic DNA containing the rmtB gene (DNA concentration: 100 ng / μL) into th...

Embodiment 3

[0056] Example 3: Comparison of detection sensitivity between rmtB gene LAMP detection method and common PCR method

[0057] Get the Escherichia coli genomic DNA (concentration is 500ng / μL) containing rmtB gene, and dilute it by 10 times, which is 10% of the stock solution respectively. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 times, 1 μL was used as a template for LAMP reaction and PCR reaction respectively. Add 1 μL of the bacterial genomic DNA containing the rmtB gene diluted above to the LAMP reaction tube and the PCR reaction tube respectively.

[0058] The LAMP reaction tube was placed in a constant temperature water bath at 63°C to react for 45-60 minutes, and the color change was directly observed with the naked eye after the reaction.

[0059] The primers used in the PCR reaction were designed with the Primer5.0 software based on the conserved sequence of the rmtB gene known on NCBI, and were synthesized by a DNA synthesizer according to the ...

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Abstract

The invention discloses an LAMP detection primer, detection kit and detection method for 16S rRNA methylase rmtB. The detection primer disclosed by the invention consists of a pair of outer primers F3 and B3, a pair of inner primers FIP and BIP, and a ring primer L. The detection kit comprises a plurality of LAMP reaction tubes loaded with reaction liquid, and the reaction liquid contains 2* constant-temperature reaction buffer liquid, Bst DNA polymerase, primers and the like. The detection method comprises the following steps: firstly extracting DNA of bacterial genomes to be detected, adding the extracted DNA to the reaction liquid of the LAMP reaction tubes, performing a constant-temperature reaction in a water bath of 63 DEG C for 45-60 minutes, and judging the result according to the phenomenon whether white precipitate exists or not or whether the reaction liquid becomes green or not after calcein is added. According to the LAMP detection primer, detection kit and detection method disclosed by the invention, the problems that the conventional PCR technique is long in time, large in workload, large in cross contamination and complex in operation are solved, and the LAMP detection primer, the detection kit and the detection method have the advantages of strong specificity, high sensitivity and high rapidness, and low cost, are simple to operate, and are suitable for on-site rapid detection.

Description

technical field [0001] The invention belongs to the technical field of bacterial gene detection, and in particular relates to a LAMP detection primer, a detection kit and a detection method for aminoglycoside antibiotic resistance gene 16s rRNA methylase rmtB. Background technique [0002] 16S rRNA methylase is a new determinant of drug resistance reported in 2003, which mediates high-level drug resistance of bacteria to a variety of aminoglycosides. Found 10 16S rRNA methylases encoded by at least 12 alleles. This type of drug resistance determinant has a unique mechanism of action. It can use S-adenosylmethionine molecule as a methyl donor to perform post-transcriptional methylation on ribosomal RNA, so that aminoglycosides cannot be combined with their targets. , resulting in a variety of Gram-negative bacilli being highly resistant to amikacin, gentamicin and other commonly used clinical aminoglycosides. Drug resistance encoding 16S rRNA methylases such as RmtA, RmtB, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689
Inventor 刘玉庆齐静杨炜华黄保华骆延波胡明李璐璐张庆
Owner RECOM QINGDAO BIOTECH CO LTD
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