Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene

A loop-mediated isothermal and multi-drug resistance technology, which is applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., to achieve the effects of shortened time, high sensitivity and strong specificity

Active Publication Date: 2011-11-02
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 multi-drug resistance cfr Gene blank, the present invention provides primers for multi-drug resistance cfr gene using loop-mediated isothermal amplification

Method used

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  • Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene
  • Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene
  • Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation for detection of multidrug resistance cfr Gene Kit

[0042] Set up to detect multidrug resistance cfr The kit of gene, this kit comprises the LAMP reaction tube that reaction liquid is housed,

[0043] 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 The loop primer L and the balance are sterilized double distilled water;

[0044] Wherein, the activity unit of Bst DNA polymerase is 8U / μL;

[0045]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.

[0046] The nucleotide sequences of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, and loop primer L are as follows:

[0047] Outer primer F3: 5'-TCTTGAGCATGCAAACGA-3',

[00...

Embodiment 2

[0056] Example 2: Detection of multidrug resistance with loop-mediated isothermal amplification cfr genetic testing methods

[0057] (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.

[0058] (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. In each system, there are detection tubes, a positive control tube and a negative control tube. Add 2 μL of the DNA sample to be tested in the dete...

Embodiment 3

[0062] Example 3: Multidrug Resistance cfr Comparison of detection sensitivity between gene LAMP detection method and common PCR method

[0063] Take and contain cfr Genetic Staphylococcus squirrel bacterial genomic DNA (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. That is, tubes 1 to 7 in the LAMP reaction and the PCR reaction were respectively added with the above doubling-diluted containing cfr Genomic bacterial genomic DNA 1 μL, add 1 μL of sterilized double distilled water to tube 8.

[0064] The LAMP reaction was placed in a constant temperature water bath at 63°C for 35 min. After the reaction, the reaction tube without the fluorescent dye calcein was directly observed with the naked eye for the formation of white precipitate, and the reacti...

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Abstract

The invention belongs to the technical field of bacterial gene detection and provides primers of loop-mediated isothermal amplification (LAMP) for detecting a multidrug-resistant cfr gene, which include a pair of outer primers, a pair of inner primers and a loop primer, wherein the molar ratio of the outer primers to the inner primers is 1:8, and the molar ratio of the outer primers to the loop primer is 1:4. The invention also provides a kit for detecting the multidrug-resistant cfr gene; and the kit comprises a plurality of LAMP tubes each of which has a reaction liquid composed of an isothermal reaction buffer solution, BstDNA polymerase, an inner primer FIP, an inner primer BIP, an outer primer F3, an outer primer B3, a loop primer L and sterile double-distilled water. The invention further provides a method for detecting the multidrug-resistant cfr gene, which comprises the following steps of: extracting the genomic DNA of a bacterial strain to be detected and detecting by using the kit under such a condition that the amplification reaction is carried out for 35 minutes in a water bath of 63 DEG C. Based on the LAMP technique, the specificity is enhanced and higher than that of the PCR detection method, the detection time is greatly reduced and the detection is simple and rapid.

Description

technical field [0001] The invention belongs to the technical field of bacterial gene detection, in particular to loop-mediated isothermal amplification multi-drug resistance cfr Gene primers also involve a loop-mediated isothermal amplification method for detection of multidrug resistance cfr Genetic kit, also related to detection of multidrug resistance by loop-mediated isothermal amplification method cfr genetic testing methods. Background technique [0002] In recent years, due to the wide application and irrational use of broad-spectrum antibiotics, the drug resistance of bacteria has become stronger and stronger, and a large number of multi-drug resistant strains (MDRO) have emerged. Currently common ones include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), bacteria producing extended-spectrum β-lactamases (ESBLs) (such as Escherichia coli, Klebsiella, Stenotrophomonas maltophilia, etc.) and multidrug-resistant Aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02C12N15/11
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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