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Liquid chip primer group for multi-drug resistance gene detection and application of liquid chip primer group

A drug-resistant gene and liquid chip technology, applied in recombinant DNA technology, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as poor timeliness, many influencing factors, and long detection cycle

Active Publication Date: 2021-04-27
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, clinical laboratories mostly use drug susceptibility testing to detect the phenotype of drug-resistant bacteria and conventional PCR to detect the genotype, but the detection cycle is long, the timeliness is poor, the detection throughput is low, and there are many influencing factors. Requirements for high sensitivity and timely feedback

Method used

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  • Liquid chip primer group for multi-drug resistance gene detection and application of liquid chip primer group
  • Liquid chip primer group for multi-drug resistance gene detection and application of liquid chip primer group
  • Liquid chip primer group for multi-drug resistance gene detection and application of liquid chip primer group

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Primer screening for detection of multiple drug resistance genes, select qnrS NG_050543.1, aac(6')-Ib-cr NG_052123.1, gyrA MF741921.1, sul-1 NG_048082.1, sul-2 NG_048106.1, sul in GenBank -3 NG_048120.1, aadA1 NG_047324.1, Aph(3')-Ⅱ-a NG_047417.1 and tetB NG_048170.1 were used as reference sequences to compare sequence homology, select their conserved regions, and design by primerplexP2273 multiple primer software Candidate primers, and optimized through a large number of reaction conditions, comparative tests and verification tests to obtain the liquid chip primer set for multiple drug resistance gene detection; the primer set is synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; the primer set The structure of the upstream primers is F1-F2-F3, where the sequence of F1 is reverse complementary to the sequence connected to the compatible microsphere used for reading the results of the liquid chip, and the sequence of F3 is the specific gene sequence of the drug-r...

Embodiment 2

[0083] specificity test

[0084] Sample DNA Extraction

[0085] (1) Extraction by boiling method: Take 1 mL of cultured medium (Escherichia coli) and centrifuge at 12,000 rpm for 5 min, wash the precipitate once with 500 μL sterile saline, resuspend with 100 μL sterile ultrapure water, and boil for 10 min. Cool in ice for 10 minutes, centrifuge at 12000 rpm for 5 minutes to take the supernatant.

[0086] (2) Bacterial Genomic DNA Mini Kit Extraction.

[0087] The present invention selects qnrS NG_050543.1, aac(6')-Ib-cr NG_052123.1, gyrA MF741921.1, sul-1 NG_048082.1, sul-2 NG_048106.1, sul-3 NG_048120.1, aadA1NG_047324.1, Aph(3')-Ⅱ-a NG_047417.1 and tetB NG_048170.1 were used as reference sequences, and primers were redesigned to amplify the gene sequence larger than the above-mentioned amplified target fragment. The sequence length was 500bp~1000bp, and ligated into pMD18 -T vector, transformed into Escherichia coli DH5α. After the positive clones were picked and enriche...

Embodiment 3

[0099] Sensitivity test

[0100] Select the positive plasmids of qnrS, aac(6')-Ib-cr, gyrA, sul-1, sul-2, sul-3, aadA1, Aph(3')-II-a and tetB and their corresponding primers, and configure them as Multiplex PCR reaction system: 2×PROMEGA Go Taq GreenMaster Mix 25 μL; the working concentration of each primer is 10 μM, take qnrS-F 0.8 μL, qnrS-R 0.8 μL, aac(6’)-Ib-cr-F 1.2 μL, aac( 6')-Ib-cr-R 1.2μL, gyrA-F 0.8μL, gyrA-R 0.8μL, sul-1-F 0.8μL, sul-1-R 0.8μL, sul-2-F 1.8μL, sul- 2-R 1.8μL, sul-3-F 1.8μL, sul-3-R 1.8μL, aadA1-F 0.8μL, aadA1-R 0.8μL, Aph(3')-Ⅱ-a-F 0.6μL, Aph(3' )-Ⅱ-a-R 0.6 μL, tetB-F 1.2 μL and tetB-R 1.2 μL, template 1 μL; replenish water to 50 μL; reaction conditions were pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds , cycled 35 times; 72°C for 10min. Add 1 μL of positive plasmids for each gene according to the design in Table 4.

[0101] Table 4 Control tabl...

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Abstract

The invention relates to the field of molecular identification, in particular to a liquid chip primer group for multi-drug resistance gene detection and an application of the liquid chip primer group. When the primer group is used for performing multiplex PCR to simultaneously detect nine drug-resistant genes, the nine groups of primer pairs have no cross reaction, are good in specificity and high in sensitivity, have the advantages of high flux, high speed, low cost, high sensitivity, good specificity, good repeatability and wide linear range, and have a great prospect in the aspect of detection and application of the drug-resistant genes. After PCR amplification, a PCR product does not need to be treated and can be directly hybridized with the microspheres, so that the time is saved, and the process is simplified.

Description

technical field [0001] The invention relates to the field of molecular identification, in particular to a liquid chip primer set for detecting multiple drug resistance genes and its application. Background technique [0002] qnrS, aac(6')-Ib-cr, gyrA, sul-1, sul-2, sul-3, aadA1, Aph(3')-Ⅱ-a and tetB belong to quinolones, aminoglycosides and sulfonamides, respectively and tetracycline resistance genes, these four types of drugs are the main antibacterial drugs in both medical and veterinary use, and a large number of multidrug resistance bacteria (multidrug resistance, MDR) have been produced in the long-term use. [0003] Drug resistance genes can be transmitted between different bacteria through mobile elements such as plasmids, integrons, and transposons, and can also be transmitted between animals, humans, and the environment. The detection and monitoring of bacterial resistance is helpful to understand the prevalence and distribution of drug-resistant bacteria and drug-...

Claims

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Application Information

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IPC IPC(8): C12Q1/6837C12Q1/689C12N15/11
CPCC12Q1/6837C12Q1/689C12Q2531/113C12Q2563/107C12Q2537/143
Inventor 曲志娜韩天飞赵建梅王君玮刘俊辉王娟赵格刘娜王琳张喜悦李月华黄秀梅张青青高玉斌
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT