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Primer composition, kit and detection method for detecting OXA48 family genes by LAMP (Loop-Mediated Isothermal Amplification)

The technology of a primer composition and a kit is applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as large errors, expensive instruments, and insignificant changes in the color of hydroxynaphthol blue, etc. Achieve the effect of simple operation, high sensitivity and reasonable design

Pending Publication Date: 2022-04-15
CHENGDE MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If some scholars use fluorescent quantitative PCR instrument for LAMP reaction, they can record the curve in real time and perform qualitative and semi-quantitative detection, but the instrument is expensive and requires professionals to operate the instrument; some scholars also use hydroxynaphthol blue as the chromogenic agent of LAMP , the reaction is about 1 hour, but the color change of hydroxynaphthol blue is not obvious, and different people observe with naked eyes, and the error is large

Method used

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  • Primer composition, kit and detection method for detecting OXA48 family genes by LAMP (Loop-Mediated Isothermal Amplification)
  • Primer composition, kit and detection method for detecting OXA48 family genes by LAMP (Loop-Mediated Isothermal Amplification)
  • Primer composition, kit and detection method for detecting OXA48 family genes by LAMP (Loop-Mediated Isothermal Amplification)

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Experimental program
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Effect test

Embodiment 1

[0038] A LAMP kit for detecting carbapenemase-resistant Enterobacter cloacae OXA48 gene, including a primer composition for detecting carbapenemase-resistant Enterobacter cloacae KPC gene by LAMP; said LAMP detects carbapenemase-resistant Enterobacter cloacae KPC gene The primer composition of Enterobacter cloacae OXA48 gene includes 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.4 μM forward loop primer LF, 0.4 μM reverse loop primer LB.

[0039] Described primer composition comprises:

[0040] Forward outer primer F3: 5'-AATAGCTTGATCGCCCTC-3';

[0041] Reverse outer primer B3: 5'-CCATAATCGAAAGCATGTAGC-3';

[0042]Forward internal primer FIP:

[0043] 5'-GATTCCAAGTGGCGATATCGCGGCGTGGTTAAGGATGAAC-3';

[0044] Reverse inner primer BIP:

[0045] 5'-TAATCACCGCGATGAAATATTCAGTCTTGCTCATACGTGCCTC-3';

[0046] Forward loop primer LF: 5'-TGTCCATCCCACTTAAAGACTTG-3';

[0047] Reverse loop primer LB...

Embodiment 2

[0052] Embodiment 2: different final concentration MgSO Test

[0053] A method for LAMP detection of carbapenemase-resistant Enterobacter cloacae OXA48 gene, carried out according to the following sequence of steps:

[0054] (1) Add 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.4 μM forward loop primer, 0.4 μM reverse inner primer to the reaction tube Loop primer, 0.2M betaine, (0-12)mMMgSO4, 1.4mM dNTPs, 8U Bst DNA polymerase, 2.5μl of 10×buffer, 1μl template DNA, 0.05mM calcein, 0.6mM MnCl2, (8.6-5.6 ) μl of sterile ddH2O, and place the reaction tube in a constant temperature water bath; the temperature of the constant temperature water bath is 65°C, and the reaction time is 40 minutes;

[0055] (2) Place the reaction tube in step (1) on an ice box to terminate the reaction, and judge the amplification result according to the color change in the reaction tube.

[0056] The kit also includ...

Embodiment 3

[0064] Embodiment 3: different final concentration betaine experiments

[0065] A method for LAMP detection of carbapenemase-resistant Enterobacter cloacae OXA48 gene, carried out according to the following sequence of steps:

[0066] (1) Add 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.4 μM forward loop primer, 0.4 μM reverse inner primer to the reaction tube Loop primer, (0-1.2)M betaine, 6mMMgSO4, 1.4mM dNTPs, 8U Bst DNA polymerase, 2.5μl of 10×buffer, 1μl template DNA, 0.05mM calcein, 0.6mM MnCl2, (8.1-2.1) μl sterile ddH20, and place the reaction tube in a constant temperature water bath; the temperature of the constant temperature water bath is 65°C, and the reaction time is 40 minutes;

[0067] (2) Place the reaction tube in step (1) on the ice box to terminate the reaction, judge the amplification result according to the color change in the reaction tube, the result is as follows ...

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Abstract

The invention relates to a primer composition for detecting carbapenemase-resistant enterobacter cloacae OXA48 family genes through LAMP, a corresponding kit and a detection method, common gene segments of the OXA48 family are selected, the common gene segments comprise blaOXA-48, blaOXA-181, blaOXA-232, blaOXA-204, blaOXA-162 and blaOXA-244, primers needed in a loop-mediated isothermal amplification system are designed, and the primers are used for detecting carbapenemase-resistant enterobacter cloacae OXA48 family genes through LAMP. A reaction system, reaction conditions, coloring agent concentration and the like are repeatedly optimized, and the improved OXA48 family LAMP detection kit is established. The LAMP detection kit not only can be used for detecting OXA48 family genes of enterobacter cloacae, but also can be used for detecting other bacteria, and the bacteria containing the drug-resistant genes can be detected. The LAMP detection kit is simple and convenient to operate and low in operation risk.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and relates to a primer composition, a kit and a detection method, in particular to a primer composition and a corresponding kit for LAMP detection of carbapenemase-resistant Enterobacter cloacae OXA48 family genes and detection methods. Background technique [0002] Enterobacteriaceae is one of the common pathogenic bacteria in community and hospital infections. Since the 1980s, carbapenem antibacterial drugs have been considered to be effective against intestinal bacteria producing extended-spectrum β-lactamase (ESBLS) and AmpC enzymes. Bacillus infection is the most effective means, but in recent years, the overuse of antimicrobial drugs in clinical practice has led to the production of carbapenem-resistant Enterobacteriaceae (CRE), which has become a widespread trend worldwide. Carbapenemase is a carbapenem-hydrolyzing lactamase, mainly including KPC serine β-lactamase, NDM, IMP,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12N15/11C12R1/01
Inventor 刘金霞杨思煕任立群李冉王丽娜
Owner CHENGDE MEDICAL UNIV
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