Primers and kit for detecting macrolide drug-resistance genes of bacteria

A macrolide and drug resistance gene technology, applied in the field of microbial detection, can solve the problems of unsuitable clinical large-scale application, long time required, complicated operation, etc., and achieves simple operation, low cost and high sensitivity. Effect

Active Publication Date: 2015-04-29
CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the initial qualitative PCR and electrophoresis to the later gene chip and other technologies, it is possible to detect bacterial antibiotic-related drug resistance genes, but qualitative PCR and electrophoresis have defects such as easy contamination and cumbersome operation. Gene chip technology can detect multiple drug resistance genes at the same time , but the cost is also very high, the operation is complicated, and the time required is long, so it is not suitable for large-scale clinical application

Method used

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  • Primers and kit for detecting macrolide drug-resistance genes of bacteria
  • Primers and kit for detecting macrolide drug-resistance genes of bacteria
  • Primers and kit for detecting macrolide drug-resistance genes of bacteria

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: detect the nucleic acid sequence of 3 kinds of drug-resistant genes drug-resistant genes ermA, ermB, ermC of bacterial macrolides, and 3 pairs of primers are synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.:

[0037] Primer pairs for amplifying the ermA resistance gene:

[0038] F1 (SEQ ID NO: 1): 5'-TTGATGGAGGCTTATGTCAAGTGA-3',

[0039] R1 (SEQ ID NO: 2): 5'-TTTTCGCAAATCCCTTCTCAAC-3';

[0040] Primer pairs for amplifying the ermB drug resistance gene:

[0041] F2 (SEQ ID NO: 3): 5'-TCACCGAACACTAGGGTTGCTC-3',

[0042] R2 (SEQ ID NO: 4): 5'-CTGTGGTATGGCGGGTAAGTTT-3';

[0043]Primer pairs for amplifying the ermC drug resistance gene:

[0044] F3 (SEQ ID NO: 5): 5'-CGTAACTGCCATTGAAATAGACCA-3',

[0045] R3 (SEQ ID NO: 6): 5'-TCTTTTAGCAAACCCGTATTCCAC-3'.

Embodiment 2

[0046] Embodiment 2: the preparation method of kit.

[0047] (1) PCR reaction solution: Fast EvaGreen qPCR Master Mix (purchased from U.S. Biotium Company), is a 2*PCR reaction enzyme premix solution, which contains PCR reaction buffer solution of the present invention, DNA polymerase, EvaGreen fluorescent dye, Mg 2+ and dNTPs, stored at -20°C;

[0048] (2) Primer mixture: Submit the nucleotide sequences shown in SEQ ID NO: 1 to 6 to be synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., then mix them in a tube, dissolve them in double distilled water, and The final concentration of one primer is 1 μmol / L, and stored at -20°C;

[0049] (3) Positive control: three bacterial genomic DNAs and Escherichia coli genomic DNAs respectively containing ermA, ermB, and ermC drug-resistant genes, wherein the concentration of each bacterial genomic DNA containing drug-resistant genes was 10ng / μL, and the Escherichia coli genome The DNA concentration is 50ng / μL, stored at -20°C;

...

Embodiment 3

[0051] Embodiment 3: detection method.

[0052] Instrument: Roche LightCyclery Fluorescent quantitative PCR detector, BECKMAN Microfuge Desktop micro refrigerated centrifuge, Eppendorf 5810R desktop refrigerated centrifuge, Taicang Hualida Laboratory Equipment Company WH-866 vortex oscillator.

[0053] (1) Preparation of bacterial genomic DNA template: refer to published literature, use corresponding commercial DNA extraction kits for different types of bacterial specimens, and prepare bacterial genomic DNA according to the kit instructions, and use it as a PCR reaction template for future use.

[0054] (2) Using the genomic DNA obtained in step (1) as a template, use 3 pairs of specific primers and high-performance fluorescent dyes to perform amplification detection of bacterial macrolide drug-resistant genes ermA, ermB, and ermC, specifically including the following steps ;

[0055] (2a) Preparation of PCR reaction solution: Take out each component of the kit from the -...

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Abstract

The invention discloses primers and a kit for detecting the macrolide drug-resistance genes of bacteria, and belongs to the technical field of microbiological detection. The sequences of the primers for detecting the macrolide drug-resistance genes of bacteria provided by the invention are shown in SEQ ID NO: 1-6, and the three primer pairs are capable of specifically amplifying the three macrolide drug-resistance genes of ermA, ermB and ermC of multiple bacteria. The invention further discloses a kit for detecting the macrolide drug-resistance genes of bacteria, wherein the kit contains an EvaGreen fluorescent dye, and is capable of rapidly and accurately detecting the macrolide antibiotic-related drug-resistance genes of bacteria. The primers and the kit disclosed by the invention have the advantages of simplicity and convenience in operation, high specificity, high sensibility, low cost, high flux and the like, and can be used for rapidly detecting macrolide antibiotic-related drug-resistance genes of clinic pathological bacteria and providing a reference for clinic treatment.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a primer and a kit for detecting bacterial macrolide drug-resistant genes. Background technique [0002] Macrolide antibiotics are a class of antibacterial drugs with the basic chemical structure of a macrolide ring and a similar antibacterial spectrum. They can be further divided into 14, 15, and 16-membered rings according to the number of carbon atoms that make up the macrolide ring. Macrolide antibiotics. Macrolides include first-generation erythromycin, which replaces penicillin, and second-generation clarithromycin, roxithromycin, etc. Macrolide antibiotics are a class of rapid bacteriostatic agents. [0003] The main effect of macrolide antibiotics on bacteria is to inhibit the synthesis of bacterial proteins. Studies have shown that the nascent peptide release channel of bacterial ribosomes has a certain degree of conservation. In the riboso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/689C12Q2600/106C12Q2600/156C12Q2600/16
Inventor 任绪义虞闰六俞岳峰
Owner CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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