Diagnostic method for quickly detecting drug resistance gene CMY-2 of germ AmpC enzyme

A CMY-2 and drug-resistant gene technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of severe prevalence of plasmid AmpC enzyme, lack of detection methods, outbreaks of hospital infection, etc., and achieve high specificity and specificity Good performance and high sensitivity

Inactive Publication Date: 2015-09-30
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There have been reports of nosocomial infection outbreaks caused by strains producing CMY-2 plasmid AmpC enzyme, but due to the lack of relatively simple and easy detection methods, the actual epidemic situation of plasmid AmpC enzyme is much more serious

Method used

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  • Diagnostic method for quickly detecting drug resistance gene CMY-2 of germ AmpC enzyme
  • Diagnostic method for quickly detecting drug resistance gene CMY-2 of germ AmpC enzyme
  • Diagnostic method for quickly detecting drug resistance gene CMY-2 of germ AmpC enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 For Rapid Detection of Bacterial AmpC Enzyme Drug Resistance Gene CMY-2 Establishment of the LAMP kit

[0033] LAMP kit for rapid detection of bacterial extended-spectrum β-lactamase resistance gene PME-1, including the following components: (1) specific primer set; (2) DNA polymerase; (3) LAMP reaction solution; (4) Chromogenic reagent; (5) Positive control and negative control.

[0034] (1) Design of specific primers:

[0035] According to bacterial AmpC enzyme resistance gene CMY-2 (GenBank accession number is: (DQ173299)) Specifically designed 6 specific primers, the sequences are as follows:

[0036] Internal primer 1: 5'-CCTGGTAGATAACGGCAACGGTCGTTAATCGCACCATCAC-3' (SEQ ID No.1);

[0037] Internal primer 2: 5'- AGCCGATATCGCCAATAACCACACGTCTTACTAACCGATCCT-3' (SEQ ID No.2);

[0038] Outer primer 1: 5'- AGAACAACAGATTGCCGAT-3' (SEQ ID No.3);

[0039]Outer primer 2: 5'- GGCCAGTATTTCGTGACC-3' (SEQ ID No.4);

[0040] Loop primer 1: 5'-CGGAATAGCCTGCTCCTG-...

Embodiment 2

[0046] Example 2 Bacterial AmpC Enzyme Drug Resistance Gene CMY-2 rapid detection method

[0047] Utilize the kit of embodiment 1 to rapidly detect bacterial AmpC enzyme drug-resistant gene CMY-2 ,Specific steps are as follows:

[0048] (1) Extract template DNA: Extract bacterial genomic DNA by boiling water;

[0049] (2) Loop-mediated isothermal gene amplification reaction: 25 μL reaction system contains: inner primers 1 and 2 at a final concentration of 8 pmol / μL each, outer primers 1 and 2 at a final concentration of 1 pmol / μL each, loop primers 1 and 2 The final concentration is 4 pmol / μL, the reaction solution is 12.5 μL, the DNA polymerase is 8U, and the DNA to be tested is 50 ng. Make up to 25 μL with sterilized deionized water, then add 20 μL of sealing solution, and put the above reaction tube at 63 °C. Reaction 60min;

[0050] (3) Judgment of results: Add 1 μL of chromogen 1×SYBR Green I to the above reaction tube, and observe the color of the reaction solution...

Embodiment 3

[0051] Embodiment 3 specificity experiment

[0052] Utilize the method of embodiment 2 to identify not containing CMY-2 Gene clinical isolates were tested, and DEPC water was used as a negative control.

[0053] See the test results figure 2 . Bacterial AmpC enzyme resistance gene only CMY-2 The tube is green and the rest of the tubes are orange. The results show that the detection kit of the present invention has high specificity and can accurately identify bacterial AmpC enzyme drug resistance gene CMY-2 and other non-bacterial AmpC enzyme resistance genes CMY-2 differentiate.

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Abstract

The invention discloses an LAMP kit and method for quickly detecting drug resistance gene CMY-2 of a bacterial AmpC enzyme. The kit comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. DNAs of to-be-detected bacterial genomes are extracted respectively like SEQ ID NO. 1-6, six specific primers and DNA polymerase with strand displacement activity are adopted to amplify a DNA sample at 60-65 DEG C, and a color-developing agent is added to observe color changes in a reaction tube to judge whether the amplification occurs. The LAMP kit and method for quickly detecting drug resistance gene CMY-2 of the bacterial AmpC enzyme have the advantages of quickness, efficiency, simpleness and convenience in operation, high specificity, high sensitivity, simple and convenience in detection and suitability for field testing, and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the detection of pathogenic microorganisms, in particular to a rapid detection of bacterial AmpC enzyme drug resistance gene by using loop-mediated isothermal gene amplification technology (LAMP technology) CMY-2 kits and detection methods. Background technique [0002] AmpC enzyme is one of the most important β-lactamases produced by β-lactam antibacterial drug-resistant Gram-negative bacteria, and belongs to type I β-lactamase according to Bush classification (also known as inducible enzyme or C-type cephalosporin) Gram-negative bacteria (especially Enterobacter cloacae) are resistant to 1-3 generation cephalosporins, monocyclic β-lactamases, cephamycins and compound preparations containing enzyme inhibitors important reason. According to the mediation mode of transmission, it can be divided into two types: plasmid-mediated transmission and chromosome-mediated transmission. The transmissibility, high expression and persist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689
Inventor 田国宝黄曦钟兰兰张雪飞
Owner SUN YAT SEN UNIV
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