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LAMP primer combination for detecting seven types of drug-resistant genes resisting carbapenem antibiotics and application of LAMP primer combination

A primer combination and drug resistance gene technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection. It can solve the problems of complex data analysis, high price, and long detection cycle.

Inactive Publication Date: 2017-05-31
CAPITALBIO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional gene identification methods are PCR method and DNA sequencing method. The traditional PCR method has a long detection cycle and usually takes 3-4 hours; Sanger DNA sequencing method requires high primer specificity and is expensive. Later data The analysis is complicated and not suitable for clinical promotion

Method used

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  • LAMP primer combination for detecting seven types of drug-resistant genes resisting carbapenem antibiotics and application of LAMP primer combination
  • LAMP primer combination for detecting seven types of drug-resistant genes resisting carbapenem antibiotics and application of LAMP primer combination
  • LAMP primer combination for detecting seven types of drug-resistant genes resisting carbapenem antibiotics and application of LAMP primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216] Embodiment 1, the preparation of kit

[0217] The kit consists of seven LAMP primer sets, each for the detection of a carbapenem resistance gene.

[0218] The primer set used to detect the drug resistance gene OXA-23 is as follows:

[0219] Outer primer F3: 5'-GAATATGTGCCAGCCTCTA-3' (sequence 1 in the sequence listing);

[0220] Outer primer B3: 5'-TCTTTTTTGCATGAGATCAAGAC-3' (sequence 2 in the sequence listing);

[0221] Internal primer FIP: 5'-CCTTTTTCTCGCCCTTCCATTTAAATTTGAATGCCCTGATCGGA-3' (sequence 3 in the sequence listing);

[0222] Internal primer BIP: 5'-TACCGCTTGGGAAAAAGACATGGTCGCGCAAGTTCCTGA-3' (sequence 4 in the sequence listing);

[0223] Loop primer LF: 5'-CCGTTTTCTGGTTTCCAA-3' (sequence 5 in the sequence listing);

[0224] Loop primer LB: 5'-ACACTAGGAGAAGCCATGA-3' (sequence 6 in the sequence listing).

[0225]The primer set used to detect the drug resistance gene OXA-24 is as follows:

[0226] Outer primer F3: 5'-CTGTGTTCCAATATTTGTATTTCC-3' (sequence ...

Embodiment 2

[0268] Embodiment 2, specificity experiment

[0269] Test sample 1: a plasmid containing the drug resistance gene OXA-23. The preparation method of the plasmid containing the drug-resistant gene OXA-23 is as follows: The DNA fragment between EcoRI and SpeI of the -T Easy plasmid was replaced with the DNA molecule shown in the nucleotide sequence of Genebank No. KP203815.1, and the resulting recombinant plasmid was a plasmid containing the drug resistance gene OXA-23.

[0270] Test sample 2: a plasmid containing the drug resistance gene OXA-24. The preparation method of the plasmid containing the drug-resistant gene OXA-24 is as follows: The DNA fragment between EcoRI and SpeI of the -T Easy plasmid was replaced with the DNA molecule shown in the nucleotide sequence of Genebank No. KR922888.1, and the resulting recombinant plasmid was a plasmid containing the drug resistance gene OXA-24.

[0271] Test sample 3: a plasmid containing the drug resistance gene OXA-58. The pre...

Embodiment 3

[0290] Embodiment 3, sensitivity experiment

[0291] Test sample 1: the plasmid containing the drug resistance gene OXA-23 prepared in Example 2.

[0292] Test sample 2: the plasmid containing the drug resistance gene OXA-24 prepared in Example 2.

[0293] Test sample 3: the plasmid containing the drug resistance gene OXA-58 prepared in Example 2.

[0294] Test sample 4: the plasmid containing the drug resistance gene OXA-66 prepared in Example 2.

[0295] Test sample 5: the plasmid containing the drug resistance gene KPC-2 prepared in Example 2.

[0296] Test sample 6: the plasmid containing the drug resistance gene IMP-4 prepared in Example 2.

[0297] Test sample 7: the plasmid containing the drug resistance gene VIM-2 prepared in Example 2.

[0298] 1. Extract the plasmid DNA of the sample to be tested, and perform gradient dilution with sterile water to obtain each dilution.

[0299] 2. Using the dilution obtained in step 1 as a template, the primer sets prepared in ...

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Abstract

The invention discloses an LAMP primer combination for detecting seven types of drug-resistant genes resisting carbapenem antibiotics and application of the LAMP primer combination. The invention first provides a primer combination consisting of 42 types of DNA molecules as shown in sequences 1-42. The primer combinations can be applied to detect whether a to-be-tested bacterium or sample contains a drug-resistant gene OXA-23 and / or a drug-resistant gene OXA-24 and / or a drug-resistant gene OXA-58 and / or a drug-resistant gene OXA-66 and / or a drug-resistant gene KPC-2 and / or a drug-resistant gene IMP-4 and / or a drug-resistant gene VIM-2. The primer combination provided by the invention is identified to be used for detecting the seven types of drug-resistant genes resisting carbapenem antibiotics, has high specificity and high sensitivity, and can implement simple, convenient, quick and accurate detection. The LAMP primer combination provided by the invention has a significant promotion value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of LAMP primers for detecting seven drug-resistant genes resistant to carbapenem antibiotics and an application thereof. Background technique [0002] my country is one of the countries where the abuse of antibiotics is the most serious. However, with the increase in the use of antibiotics, bacterial drug resistance is also becoming more and more serious. There are many kinds of antibiotics with various mechanisms of action, among which carbapenems in β-lactam antibiotics can inhibit bacterial cell wall Synthetic to play a bactericidal effect. Drug-resistant genes in drug-resistant bacteria (such as drug-resistant gene OXA-23, drug-resistant gene OXA-24, drug-resistant gene OXA-58, drug-resistant gene OXA-66, drug-resistant gene KPC-2, drug-resistant gene IMP- 4 or drug-resistant gene VIM-2) can complete the production of carbapenem antibiotics by producin...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
CPCC12Q1/689C12Q1/6844C12Q2600/16C12Q2531/119C12Q2537/1376C12Q2563/107
Inventor 张岩刘莹莹王颢婷邢婉丽程京
Owner CAPITALBIO CORP
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