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Gene for mediating drug resistance of vancomycin and application thereof

A technology of drug resistance gene and vancomycin, which is applied in the field of biomedicine, can solve the problems of missed diagnosis of infection cases, undetectable drug resistance genes, mistreatment, etc., and achieves the effect of simple operation.

Inactive Publication Date: 2012-07-11
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular biological detection methods such as PCR and gene chips in the prior art, which take drug-resistant genes as detection targets, have the advantages of convenience, quickness and high specificity, but the establishment of such methods needs to be based on the DNA sequence of known drug-resistant genes. The premise is that some new drug-resistant genes are often undetectable due to lack of DNA sequences, leading to missed diagnosis and mistreatment of infected cases, resulting in serious consequences

Method used

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  • Gene for mediating drug resistance of vancomycin and application thereof
  • Gene for mediating drug resistance of vancomycin and application thereof
  • Gene for mediating drug resistance of vancomycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Enterococcus faecium with a vancomycin-resistant phenotype (Efm-HS0661, deposit number: CGMCC No.2370) was obtained by conventional identification and drug-sensitivity methods, and the universal primer vanF of the vancomycin-resistant gene was used: 5'-GGGCTGCGATATTCAAAGCTC -3; vanR: 5'-GCACACCCGACCTCACAGC-3' (patent application number: 200410067356.3) PCR amplification has the expected 294bp size band (figure 1 ); with vanA and vanB gene-specific probes vanAN: 5'-NH2-poly(T)16-GCGCGTTCAGGCTCATC-3' and vanBN: 5'-NH2-poly(T)16-GCGGGCATCGCCGTTC-3' (patent application number: 200410067356.3) drug-resistant gene detection chip for detection, did not find the existence of the two genes described; by sequencing analysis of the PCR product, a nucleotide sequence different from the known vancomycin-resistant gene was obtained ( figure 2 ), and further used TaKaRa GenomeWalking Kit (Dalian Bao Biological Company) to carry out PCR product wing sequence sequencing and bioinformati...

Embodiment 2

[0036] DNAStar bioinformatics analysis software was used to compare the new gene sequence with other vancomycin-resistant genes vanA (gene accession number: X56895), vanB (gene accession number: AF192329), vanD (gene accession number: AB183866) and vanG (gene Accession number: DQ212986) for sequence comparison, the unique sequence of the new gene was clarified, and according to the sequence difference between the new gene sequence and the known drug-resistant gene, the universal primer vanF for the vancomycin-resistant gene: 5'- GGGCTGCGATATTCAAAGCTC-3 and vanR: 5'-GCACACCCGACCTCACAGC-3' were optimized, and degenerate bases were introduced at the 3' end of the primers. The optimized universal primers were: NvanF: 5'-GGGCTGCGATATTCAA AGHTC-3; and NvanR: 5 '-GCACACCCGACCTCACAVCC-3'.

[0037] A TaqMan probe 5′FAM-TTCGG CGGTTTGTATGGACAAATCAC-3′TAMERA was designed according to a unique sequence located between the universal primers NvanF and NvanR in the above-mentioned new gene, a...

Embodiment 3

[0039] Referring to "Drug-resistant bacteria detection chip and its preparation method and application method" (patent application number: 200410067356.3), the 5' end of the vancomycin-resistant downstream primer vanR was labeled with Cy-5 fluorescein, and combined with the upstream primer vanR for amplification For samples containing vanA, vanB and new vancomycin-resistant genes, the obtained PCR products can be labeled with Cy-5 fluorescein; design vancomycin-resistant gene oligonucleotides according to the shared conserved sequences of vanA, vanB and the new genes of the present invention General-purpose chip detection probe: NH2-poly(T)10-GGCTGYGAYATTCAAAGYTCNGYRRTTTGYATGGA, according to the unique sequence design of vancomycin-resistant new gene of the present invention, oligonucleotide-specific chip detection probe: NH2-poly(T)10-CACTGGCAT ATATTATTGCGAA( The nucleotide sequence of the probe is arranged in the direction of 5'>3'; poly(T)10 is added at the 5' end to reduce ...

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Abstract

The invention belongs to the technical field of biomedicine, and relates to a gene for mediating drug resistance of vancomycin and application thereof. The drug-resistant gene has a nucleotide sequence of SET ID.1, and protein encoded by the gene has an amino acid sequence of SEQ ID.2. The obtained gene sequence is a novel drug-resistance deciding gene of vancomycin resistant Enterococcus, and can prepare probe sequences of PCR amplification and gene microarray diagnostic probes for clinical diagnosis of resisting infection of the vncomycin resistant Enterococcus. The invention provides a novel detection target of drug-resistant strains for clinical practice, and provides a method for detecting Enterococcus carrying novel vancomycin resistance gene by Real-time PCR and a method for detecting the Enterococcus carrying the novel vancomycin resistance gene by a gene microarray; and the detection methods have the advantages of quickness, accuracy, simple and convenient operation, and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a gene mediating vancomycin resistance, in particular to a DNA sequence of a new gene causing Enterococcus faecium to resist vancomycin and its application. Background technique [0002] Enterococcus spp. is part of the normal flora of the human intestinal tract and genitourinary tract. It can also be used as an opportunistic pathogen to cause infection of various organs and tissues in the human body. Among the infections caused by Enterococcus, urinary tract infection is the most common. Common, can also cause abdominal cavity, pelvic cavity, wound infection, sepsis, endocarditis and other serious infections. According to related reports, Enterococcus infection has been on the rise in recent years, and has become one of the main pathogens of nosocomial infections, ranking third among the pathogens causing bloodstream infections such as sepsis. Enterococcus bacteria mainly incl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/315C12Q1/68C12N1/20C12R1/01
Inventor 徐晓刚王明贵林东昉胡付品吴湜叶信予郭燕刘杨朱德妹张婴元
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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