LAMP method for detecting bacterial resistance to florfenicol

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...

AI Technical Summary

Problems solved by technology

These two detection methods consume a lot of experimental consumables and perform tedious experimental operations, which are time-consuming and labor-intensive. The test results are mainly judged by naked eyes, which increases human factors.

[0004] The protein encoded by the bacterial florfenicol resistance gene floR is an export pump protein, which can specifically pump florfenicol in bacteria out of the body, so that florfenicol cannot exert its function of inhibiting bacterial protein synthesis , losing its ability to inhibit or kill bacteria

At present, conventional PCR method is used for the detection of bacterial florfenicol resistance gene floR. This method has high accuracy, but requires complex equipment, time-consuming, high cost, insufficient sensitivity and specificity, and easy amplification of products. Contamination of the PCR instrument is not suitable for large-scale detection in epidemiological investigations

[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

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