LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene as well as kit and method for detecting superbacteria NDM-1 gene

A loop-mediated isothermal, super-bacteria technology, applied in biochemical equipment and methods, microbial assay/inspection, DNA/RNA fragments, etc., to achieve the effects of rapid cost, simple operation method and strong specificity

Active Publication Date: 2011-11-16
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]In order to solve the problems of PCR amplification prone to false positives, high detection cost, complicated operation, and long detection time, the use of LAMP to detect new superbugs NDM- 1 gene blank, the present invention provides primers for isothermally amplifying the superbug NDM-1 gene

Method used

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  • LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene as well as kit and method for detecting superbacteria NDM-1 gene
  • LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene as well as kit and method for detecting superbacteria NDM-1 gene
  • LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene as well as kit and method for detecting superbacteria NDM-1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Preparation detects super bacteria NDM-1 Gene Kit

[0043] Set up to detect superbugs NDM-1 The kit of gene, this kit comprises the LAMP reaction tube that reaction liquid is housed,

[0044]The reaction solution was calculated as 25 μL, containing 12.5 μL 2× isothermal reaction buffer, 1.0 μL Bst DNA polymerase, 40 pmol internal primer FIP, 40 pmol internal primer BIP, 5 pmol external primer F3, 5 pmol external primer B3, 20 pmol Loop primer LF, 20 pmol loop primer LB, the balance is sterile double distilled water;

[0045] Among them, the activity unit of Bst DNA polymerase is 8U / μL;

[0046] 2× isothermal reaction buffer containing 40 mM Tris-HCl pH 8.8, 20 mM KCl, 16 mM MgSO 4 , 20 mM (NH4) 2 SO 4 , 0.2wt% Tween20, 1.6 M betaine, 2.8 mM dNTP.

[0047] The nucleotide sequences of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LB are as follows:

[0048] Outer primer F3: 5'-GGCCACACCAGTGA...

Embodiment 2

[0058] Example 2: Detection of superbugs by loop-mediated isothermal amplification method NDM-1 genetic testing methods

[0059] (1) Extract the total genomic DNA of the clinical strains to be tested: After culturing the clinical strains to be tested overnight, take 1 mL of the bacterial solution and centrifuge at 12,000 rpm for 2 min, discard the supernatant completely, and use the bacterial genomic DNA extraction kit ( Tiangen Biochemical Technology Beijing Co., Ltd.) extracted the total DNA of the bacterial genome, and finally dissolved it in 50 μL sterilized double-distilled water, determined the DNA concentration to be 300 ng / μL, and stored it at -20 °C for future use.

[0060] (2) LAMP amplification: using the kit in Example 1, set up two sets of reaction systems, one with fluorescent dye added and the other without. One test tube, one positive control tube and one negative control tube are set in each system. Add 2 μL of the DNA sample to be tested in the detection...

Embodiment 3

[0064] Example 3: superbugs NDM-1 Comparison of detection sensitivity between gene LAMP detection method and common PCR method

[0065] Take and contain NDM-1 Genomic DNA of the superbug Acinetobacter lophia (concentration: 300 ng / μL), diluted by 10 times, respectively 1, 10 of the stock solution -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , take 1 μL respectively as templates for LAMP reaction and PCR reaction, and sterilized double distilled water as negative control. In 7 LAMP reaction tubes and 7 PCR reaction tubes, add the above doubly diluted NDM Add 1 μL of bacterial genomic DNA of the -1 gene, and add 1 μL of sterilized double-distilled water to the eighth tube.

[0066] Among them, the LAMP reaction tube was placed in a constant temperature water bath at 65°C for 35 minutes, and after the reaction was completed, the white precipitate was directly observed with the naked eye.

[0067] The primers used in the PCR reaction are known according to NCBI NDM-1...

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Abstract

The invention belongs to the technical field of bacteria gene detection, relating to a primer of an LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene, wherein the primer consists of a pair of external primers, a pair of interior primers and a pair of loop primers, the molar ratio of the external primers to the interior primers is 1:8, and the molar ratio of the external primers to the loop primers is 1:4. A kit for detecting the superbacteria NDM-1 gene comprises a plurality of LAMP reaction tubes containing reaction solutions, each reaction solution comprises a 2* isothermal reaction buffer solution, BstDNA polymerase, an interior primer FIP, an interior primer BIP, an external primer F3, an external primer B3, a loop primer LF, aloop primer LB and sterilized double-distilled water. The invention further discloses a method for detecting the superbacteria NDM-1 gene by using the kit. Defects of long time, large work load, cross-contamination, complex operation and the like in the prior art are overcome; and the invention has the advantages of strong specificity, high sensitivity, fastness, low cost, more simpler operation method and applicability in field fast detection.

Description

technical field [0001] The invention belongs to the technical field of bacterial gene detection, in particular to loop-mediated isothermal amplification superbugs NDM-1 Primers for genes also involved in the detection of superbugs by a loop-mediated isothermal amplification method NDM-1 Genetic kit, also related to the detection of superbugs by the loop-mediated isothermal amplification method NDM-1 genetic testing methods. Background technique [0002] Super bacteria is a collective term for a class of drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus MRSA, vancomycin-resistant Enterococcus faecalis VRE, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Drug-resistant Acinetobacter baumannii, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant other Enterobacteriaceae, etc. [0003] The super bacteria NDM-1, which has recently attracted the attention of the world, is a new type of super bacteria (full name New ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 齐静刘玉庆杜以军白华骆延波朱晓玲胡明胡新新张秀美
Owner RECOM QINGDAO BIOTECH CO LTD
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