A multiple liquid-phase gene chip primer, kit and analysis method for simultaneously detecting seven aminoglycosamine drug-resistant genes

A liquid-phase gene chip and drug-resistant gene technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problem of detection of aminoglycoside drug-resistant genes by multiple immunofluorescence analysis methods , Difficult to meet detection requirements, high risk of missed detection, etc., to achieve the effect of ensuring renaturation temperature and hybridization efficiency, less sample consumption, and reducing detection costs

Active Publication Date: 2021-10-22
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported multiplex PCR for Enterococcus detects aac(6)-Ie-aph(2”)-Ia, aph(2”)-Ib, aph(2”)-Ic, aph(2”)-Id, aph(3')-IIIaa and ant(4')-Ia 6 drug-resistant genes, the size of the product fragments ranges from 71bp to 867bp, the concentration of primers used is as low as 5pmol, and the sensitivity of multiplex PCR has not been discussed, there are High risk of missed detection
Moreover, whether it is a single-plex or multiplex PCR technique, gel electrophoresis is required to determine the results, which is time-consuming and labor-intensive. In practical applications, it is difficult to meet the detection requirements when the sample volume is large.
[0006] In addition, as more and more drug resistance genes are discovered, there is no detection method that can detect seven aminoglycoside resistance genes at the same time in China, and there is no application of multiple immunofluorescence analysis to detect aminoglycosides. Reports of Glycoside Resistance Genes

Method used

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  • A multiple liquid-phase gene chip primer, kit and analysis method for simultaneously detecting seven aminoglycosamine drug-resistant genes
  • A multiple liquid-phase gene chip primer, kit and analysis method for simultaneously detecting seven aminoglycosamine drug-resistant genes
  • A multiple liquid-phase gene chip primer, kit and analysis method for simultaneously detecting seven aminoglycosamine drug-resistant genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Primer Design

[0070] After screening a large number of designed primers, it was found that the primer pairs P1 and P2, P3 and P4, P5 and P6, P7 and P8, P9 and P10, P11 and P12, P13 and P14 multiplex liquid-phase gene chip simultaneously detected 7 The drug-resistant gene works best, and its base sequence is shown below.

[0071] Primer P1: AGAGCAATAAGGGCATACCAAA (SEQ ID NO: 1),

[0072] Primer P2: CCACACTATCATAACCACTACCGA (SEQ ID NO: 2);

[0073] Primer P3: AGACGGAAAAGCCCCGAAGA (SEQ ID NO: 3),

[0074] Primer P4: GCAGAAGGCAATGTCATACCA (SEQ ID NO: 4);

[0075] Primer P5: AATCGGTAGAAGCCCCAAAAGT (SEQ ID NO: 5),

[0076] Primer P6: TGGTAGAAATGTTGTCGGTCC (SEQ ID NO: 6);

[0077] Primer P7: TGGTTCAGCAGTAAATGGTGGT (SEQ ID NO: 7),

[0078] Primer P8: ATTGCCAAGGGACAACTTCACT (SEQ ID NO: 8);

[0079] Primer P9: AGAAACTTTTGTCCACCTACCG (SEQ ID NO: 9),

[0080] Primer P10: AATGAAGCCTTTTCCGCCA (SEQ ID NO: 10);

[0081] Primer P11: AGCCGTTTCTGTAATGAAGGAG (SEQ ID NO...

Embodiment 2

[0094] Example 2: Multiple liquid-phase gene chip analysis kit for simultaneous detection of 7 pairs of aminoglycosamine resistance genes

[0095] The kit includes the following components:

[0096] (1) The primers designed in Example 1 for multiple liquid-phase gene chip analysis;

[0097] (2) 7 kinds of fluorescently encoded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the multiple liquid gene chip analysis primers; 7 kinds of microspheres All were purchased from luminex company, wherein aac(6')-Ie-aph(2")-Ia, aph(3')-IIIa, ant(4')-Ia, ant(9)-Ia, aadE, aph( 3')-Ia and aph(3")-Ib correspond to the numbers of fluorescently encoded microspheres respectively MTAG-A030, MTAG-A020, MTAG-A028, MTAG-A046, MTAG-A018, MTAG-A015 and MTAG-022.

[0098] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0099] Example 3 Establishment of a multiple liquid-phase gene chip analysis method for simultaneous detection of 7 pairs of aminoglycoside drug-resistant genes

[0100] (1) Construction of plasmid

[0101]Bacterial genomes were extracted with Tiangen’s bacterial genome extraction kit, and PCR amplification was performed with primer pairs P1 and P2, P3 and P4, P5 and P6, P7 and P8, P9 and P10, P11 and P12, P13 and P14, respectively. The amplified products were detected by agarose gel electrophoresis and purified by gel cutting. Then use Tiangen’s kit to connect the purified cDNA to the pMD-19T vector, transform the ligated product into DH5a competent cells, select a single clone, carry out bacterial liquid PCR identification, and carry out plasmid extraction on the colonies identified as positive bacteria. Mention, send for sequencing.

[0102] (2) Plasmid PCR amplification

[0103] Use the primers described in Example 1 to perform single-plex amplification on the plasmids ...

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Abstract

The invention discloses a multiple liquid-phase gene chip primer, a kit and an analysis method for simultaneously detecting seven aminoglycoside drug-resistant genes; (6')-Ie-aph(2")-Ia, aph(3')-IIIa, ant(4')-Ia, ant(9)-Ia, aadE, aph(3')-Ia and aph( 3")-Ib, and has high sensitivity and high accuracy, which can realize the simultaneous detection of multiple different target molecules in the same sample. Primers, kits and analysis methods; its technical scheme includes primer nucleotide sequences such as SEQ ID NO: 1-The primer shown in SEQ ID NO: 4; the invention belongs to the field of pathogen detection of experimental animals.

Description

technical field [0001] The invention belongs to the field of pathogen detection of experimental animals, in particular to a simultaneous detection of aminoglycosamine drug-resistant genes aac(6')-Ie-aph(2")-Ia, aph(3')-IIIa, ant(4 ')-Ia, aadE, ant(9)-Ia, aph(3')-Ia and aph(3")-Ib multiplex liquid-phase gene chip analysis method and kit. Background technique [0002] Aminoglycoside antibiotics are a class of broad-spectrum, high-efficiency antibacterial drugs, widely used in clinical practice, and are an important choice for the clinical treatment of severe infections caused by Gram-negative bacilli. They are often combined with other antibacterial drugs to treat bacterial infections. These antibiotics specifically bind to highly conserved regions of bacterial ribosomal subunits, causing changes in ribosome structure, reducing the fidelity of protein translation, inhibiting bacterial protein synthesis, and destroying cell integrity, thereby causing bacterial death. With the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6837
CPCC12Q1/6837C12Q1/6883C12Q2563/107C12Q2563/149C12Q2537/143
Inventor 徐凤姣郭鹏举黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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