LAMP primer set, kit and detection method for IMP resistance gene resistant to carbapenem antibiotics

A carbapenem, drug resistance gene technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve problems such as contamination of amplification products, achieve good specificity and strengthen detection Domain capabilities and results can be observed in real time

Active Publication Date: 2020-04-03
BEIJING TSINGHUA CHANGGUNG HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, the application number is 201510353287.0, and the title of the invention is a Chinese patent application for a diagnostic method for detecting bacterial metallo-beta-lactamase resistance gene IMP, which records a method for rapid detection of bacterial metallo-beta-lactamase resistance gene IMP, It needs to add a chromogenic agent after the reaction amplification to observe the color change in the reaction tube to judge whether it is amplified or not. Such an operation may cause unnecessary contamination of the amplified product

Method used

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  • LAMP primer set, kit and detection method for IMP resistance gene resistant to carbapenem antibiotics
  • LAMP primer set, kit and detection method for IMP resistance gene resistant to carbapenem antibiotics
  • LAMP primer set, kit and detection method for IMP resistance gene resistant to carbapenem antibiotics

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Through design, screening and verification, a LAMP primer set for IMP resistance genes resistant to carbapenem antibiotics was obtained, including the following:

[0050] 1. Determine the detection gene and sequence

[0051] Through literature research and NCBI database search, it was determined that there are 69 subtypes of carbapenem-resistant IMP resistance genes, with a total length of about 740bp, high specificity, and no other similar species except for IMP resistance genes.

[0052] The sequences of all published subtypes were collected for phylogenetic tree analysis. At the same time, in order to further improve the coverage of the primers and increase the practicability of the primers in clinical experiments, the same branch or similar branches with type 1 IMP drug resistance gene as the main detection target were collected. For other subtypes of IMP drug-resistant genes on the branch, select the common conserved sequence after sequence compression to...

Embodiment 2

[0088] Embodiment 2 specificity test

[0089] Through the analysis of 1 plasmid containing the carbapenem antibiotic IMP resistance gene fragment (SEQ ID NO: 22), 5 bacterial species containing the carbapenem antibiotic IMP resistance gene and 6 other bacteria The strains were detected by LAMP, as shown in Table 2.

[0090] Plasmid DNA containing carbapenem antibiotic IMP resistance gene fragments to be tested was prepared using Kangwei Century Plasmid DNA Extraction Kit; 5 bacterial strains containing carbapenem antibiotic IMP resistance genes and 6 other Bacterial strains were prepared using the Tiangen Bacteria Genomic DNA Extraction Kit.

[0091] The results of the LAMP reaction were judged according to the following principles.

[0092] The results judged as a positive reaction were as follows: use real-time fluorescent PCR instrument to amplify, observe the real-time fluorescence curve, the reaction time is 50min, and observe the peak time of the real-time fluorescence...

Embodiment 3

[0101] Embodiment 3 sensitivity test

[0102] Inoculate the plasmid bacteria solution containing the carbapenem antibiotic IMP resistance gene fragment (SEQ ID NO:22) to be detected in the LB liquid medium containing ampicillin (concentration ratio: 1:1000), and culture normally After 16 hours, take 1 mL of the bacterial liquid cultured in LB containing ampicillin, extract the plasmid DNA according to the method of the kit instruction (Kangwei century plasmid DNA small extraction kit), and measure the concentration and purity of the plasmid DNA with a NanoDrop 2000C ultra-micro spectrophotometer . At the same time, the extracted nucleic acid DNA was quantified using the Invitrogen Qubit 2.0 Fluorescence Quantitative Instrument and the Invitrogen Qubit Quantitative Detection Kit, and the copy number was calculated. Finally, use the LAMP test method of the present invention to measure the above DNA samples.

[0103] When the copy number of the plasmid DNA containing the carbap...

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Abstract

The present invention relates to a LAMP primer set, a kit and a detection method for an IMP resistance gene resistant to carbapenem antibiotics. The LAMP primer set comprises outer primers, inner primers and loop primers. The outer primers are shown in SEQ ID NO:1-SEQ ID NO:2, the inner primers are shown in SEQ ID NO:3-SEQ ID NO:4 and the loop primers are shown in SEQ ID NO:5-SEQ ID NO:6. The provided kit comprises the LAMP primer set. The present invention also provides the method for detecting the IMP resistance gene resistant to the carbapenem antibiotics by using the provided primer set. The provided primer set is used as a LAMP constant temperature amplification primer, has high sensitivity, strong specificity, and also high repeatability, is applied to detection of the IMP resistancegene resistant to the carbapenem antibiotics, and is short in detection time and convenient for the detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set, a kit and a detection method for an IMP drug-resistant gene resistant to carbapenem antibiotics. Background technique [0002] The detection of IMP resistance genes and their pathogenic microorganisms plays an important role in guiding clinical drug use. The traditional microbial culture method and drug susceptibility test method can provide good clinical value, but due to various reasons such as time-consuming, cumbersome operation process, and expensive equipment, they have not been widely used in clinical practice. The molecular-based PCR detection platform that emerged later has been used in the detection of IMP drug-resistant genes in recent years. The detection accuracy and time-consuming of IMP drug-resistant genes have been greatly improved through PCR amplification and probe labeling. , but because the PCR instrument amplification steps used in this type ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/689C12Q1/6844C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/689C12Q1/6844C12Q2600/106C12Q2531/119C12Q2563/107Y02A50/30
Inventor 赵秀英张岩陈燕旌边素莹
Owner BEIJING TSINGHUA CHANGGUNG HOSPITAL
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