Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli

A gram-negative bacillus, carbapenemase technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem that carbapenemase gene detection is not comprehensive enough and cannot meet NDM Gene detection and other problems, to achieve the effect of high specificity, simple operation and high detection sensitivity

Active Publication Date: 2014-03-05
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is an invention in China that uses the LAMP method to detect NDM-1 (application number 20111029784.4), but because the NDM gene has multiple subtypes due to the mutation of the site, this invention cannot meet the detection of all types of the NDM gene
And there is no detection of IMP, VIM, KPC genes
Therefore, the detection of carbapenemase gene is not comprehensive enough

Method used

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  • Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli
  • Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli
  • Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Design and synthesis of LAMP detection primers for common carbapenemase genes KPC, NDM, IMP, and VIM.

[0075] (1) Primer design method:

[0076] Download the sequences of all subtypes of KPC, NDM, IMP and VIM genes on GeneBank. So far, there are 14 subtypes of KPC, namely KPC-2, KPC-3, KPC-4, KPC-5, KPC-6, KPC-7, KPC-8, KPC-9, KPC-10, KPC- 11, KPC-12, KPC-13, KPC-14, KPC-15, GeneBank serial numbers are AY034847, AF395881, FJ473382, EU400222, EU555534, EU729727, FJ234412, FJ624872, GQ140348, HM066995, HQ641421, HQ342889, JX524191, KC433553 . There are 10 subtypes of NDM, NDM-1, NDM-2, NDM-3, NDM-4, ​​NDM-5, NDM-6, NDM-7, NDM-8, NDM-9, NDM-10, corresponding The GeneBank serial numbers are AB614355, JF703135, JQ734687, JQ348841, JN104597, JN967644; JX412225, AB744718, KC999080, KF361506. There are more than 40 subtypes of IMP, of which IMP-4 is the main type reported in my country, and the corresponding GeneBank serial number is AF244145. There are 38 subtypes of VIM. The...

Embodiment 2

[0120] Establish a LAMP kit for detecting KPC, NDM, IMP and VIM genes.

[0121] 1. The LAMP kit contains reaction reagents, sterile paraffin oil, positive control and positive control. Among them, the reaction reagent contains 2× reaction buffer (40mM Tris-HCl pH 8.8, 20mM KCl, 16mM MgSO 4 , 20mM (NH 4 ) 2 SO 4 , 0.2v / v% Tween-20, 0.8M betaine, 2.8mM dNTPs), DNA Bst polymerase, primer set (inner primer, outer primer, loop primer), indicator fluorescent indicator Syto-9 or color developer SYBR-Green, sterilized double distilled water; the positive control is the positive control strain of KPC, NDM, IMP and VIM genes, that is, the DNA of the positive samples of KPC, NDM, IMP and VIM genes is verified by conventional PCR method and sequencing. The positive specimens used in the examples were GeneBank DNA with serial numbers JF431928, JN711113, KF184385 and KF255586 respectively; the negative control was sterilized double distilled water.

[0122] The primer set corresponding to e...

Embodiment 3

[0132] Evaluation of the LAMP detection kit of the present invention.

[0133] (1) Evaluation of the specificity of the LAMP kit for detecting KPC, NDM, IMP and VIM genes.

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Abstract

The invention discloses loop-mediated isothermal amplification (LAMP) primers, a kit and a detection method for detecting common carbapenemase genes of gram negative bacilli. In the LAMP primers disclosed by the invention, klebsiella pneumoniae carbapenemase (KPC) and new delhi metallo-b-lactamase (NDM) primer groups can detect all subtypes except for NDM-10; hypoxanthine nucleotide (IMP) and vimentin (VIM) primer groups can detect common subtypes at home and abroad. The LAMP kit built by the invention is applied to joint detection of KPC, NDM, IMP and VIM genes, can cover the common carbapenemase genes of non-fermentative bacteria and enterobacteriaceae, can accurately and quickly screen the common carbapenemase genes, and has great clinical significance for timely detecting and further controlling fulminant epidemic caused by propagation of the carbapenemase genes in enterobacteriaceae. The kit disclosed by the invention is high in detection sensitivity and the minimum detection limits of the KPC, NDM, IMP and VIM genes can reach 100 CFU/reaction.

Description

technical field [0001] The invention belongs to the technical field of bacterial drug resistance gene detection, in particular to LAMP primers, a kit and a detection method for detecting KPC, NDM, IMP and VIM genes. Background technique [0002] Carbapenem antibiotics are currently the atypical b-lactam antibiotics with the broadest antibacterial spectrum and the strongest antibacterial activity. Because of its stability to b-lactamase and low toxicity, this type of drug has become one of the main antibacterial drugs for the treatment of various bacterial infections. However, with the increasing clinical application of carbapenem antibiotics, bacterial resistance to carbapenem antibiotics is inevitable, and most of these strains also show pan-drug resistance. In recent years, there have been more and more reports of carbapenem-resistant non-fermenting bacteria and even Enterobacteriaceae bacteria. It is extremely important to quickly detect and prevent the prevalence of suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119Y02A50/30
Inventor 芮勇宇程灿灿郑芬孙静静
Owner SOUTHERN MEDICAL UNIVERSITY
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