Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

LAMP (loop-mediated isothermal amplification) method for detecting florfenicol resistance of bacteria

A florfenicol and drug resistance technology, applied in the field of microbial detection, can solve problems such as difficulty in eliminating interference factors, inability to determine accurate detection results, and contamination of laboratories, and achieve the effect of a simple and convenient determination method.

Inactive Publication Date: 2014-07-23
GUANGXI VETERINARY RES INST
View PDF1 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The LAMP method has the advantages of high sensitivity, good specificity, short reaction time, convenient determination of results, and no need for expensive instruments. Analysis of the final result of the LAMP reaction cannot be quantified, and there is a risk of aerosol contamination of the laboratory. Due to the lack of real-time monitoring of the reaction, it is difficult to eliminate these interference factors, and it is impossible to make an accurate judgment on the test results

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • LAMP (loop-mediated isothermal amplification) method for detecting florfenicol resistance of bacteria
  • LAMP (loop-mediated isothermal amplification) method for detecting florfenicol resistance of bacteria
  • LAMP (loop-mediated isothermal amplification) method for detecting florfenicol resistance of bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Establishment of a LAMP method for detecting bacterial resistance to florfenicol (floR gene)

[0028] 1. Preparation of materials

[0029] Florfenicol-resistant bacteria include Streptococcus, Escherichia coli, and Enterococcus faecalis (the MIC values ​​for Florfenicol are 128 μg / mL, 256 μg / mL, and 512 μg / mL, respectively); Florfenicol-sensitive bacteria include Pasteurella Bacillus standard strain ATCC21955, Pasteurella, and Streptococcus (the MIC values ​​for florfenicol are 2 μg / mL, 1 μg / mL, and 2 μg / mL, respectively) were all isolated (or purchased) and preserved by Guangxi Veterinary Research Institute. The bacterial plasmid DNA extraction kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd., and the deoxyribonucleic acid amplification kit (LAMP method, SLP206) was purchased from Beijing Lanpu Biotechnology Co., Ltd.

[0030] 2. LAMP primers

[0031] According to the bacterial florfenicol-resistant floR gene sequence in GenBank, a set...

Embodiment 2

[0052] The specificity of embodiment 2LAMP detection method

[0053] Using the recombinant plasmid pMD18-T-floR as the positive control and sterilized ultrapure water as the negative control, 3 strains of florfenicol-resistant bacteria and 3 strains of negative control bacteria were used for LAMP amplification, and the results were as follows: figure 1 As shown, the rising curve of turbidity appeared in the positive control reaction tube after 20 minutes, and the rising curve of turbidity appeared after 30 minutes in the reaction tubes of three strains of bacterial florfenicol, which was a positive result, while the three strains of negative control There was no amplification in the bacteria and water control reaction tubes, and the result of LAMP was negative.

Embodiment 3

[0054] The sensitivity of embodiment 3LAMP detection method

[0055] The initial concentration of the recombinant plasmid pMD18-T-floR was 2.36×10 2 ng / μL, 10-fold dilution for detection, the results are as follows figure 2 and image 3 As shown, the detection limit of LAMP method is about 2.36×10 -11 ng / μL, while the detection limit of PCR method is 2.36×10 -9 ng / μL. As the concentration of the sample decreases, the time required for the turbidity value of the LAMP amplification reaction to reach 0.1 increases.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) method for detecting the florfenicol resistance of bacteria through a series of research work of preparing materials, designing and synthesizing LAMP primers, extracting bacterial plasmid DNA (deoxyribonucleic acid), optimizing an LAMP reaction system and the like, and a corresponding LAMP primer group including an outer primer pair F3 and B3 and an inner primer pair FIP and BIP is designed according to sequences of florfenicol-resistant floR genes of the bacteria. For an unknown sample, the florfenicol resistance of a bacterial sample can be judged by detecting the value of the time when the turbidity value of floR gene amplification is 0.1 only. Specific and sensitive detection results prove that the florfenicol-resistant floR genes of the bacteria subjected to specific amplification by the method is high in sensitivity which is 100 times that of the florfenicol-resistant floR genes of the bacteria subjected to specific amplification by a conventional PCR method, the reaction can be monitored in real time, the copy number of the floR genes can be quantitatively detected, the detection results can be quickly and accurately obtained, and the convenience is brought to quick detection of the florfenicol resistance of the bacteria.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, in particular to a LAMP method for detecting bacterial resistance to florfenicol (floR gene). Background technique [0002] Florfenicol (Florfenicol, FF) is a fluorinated derivative of chloramphenicol, also known as fluthiamphenicol, developed by Nagabhushan of Schering-Plough in the United States, and is an antibacterial drug mainly used to treat bacterial diseases in animals . The antibacterial mechanism of florfenicol is mainly to bind to the A site on the 50S subunit of the bacterial 70S ribosome, hinder the transpeptide reaction of peptidyltransferase, inhibit the extension of the peptide chain, and thereby inhibit the synthesis of bacterial proteins. Florfenicol is widely used in veterinary clinics to prevent and treat animal bacterial diseases because of its advantages of broad antibacterial spectrum, good absorption, wide distribution in the body, and no potential side ef...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/18C12N15/11
CPCC12Q1/6851C12Q2531/119C12Q2537/16C12Q2563/107
Inventor 李军曾芸冯世文杨威陈泽祥潘艳刘丽娅彭昊许力干马春霞胡帅禤雄标柳峰谢永平谢宇舟钟舒红
Owner GUANGXI VETERINARY RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com