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Gene for mediating drug resistance of vancomycin and application thereof

A vancomycin and drug-resistant technology, applied in the field of biomedicine, can solve the problems of missed diagnosis of infection cases, undetectable drug-resistant genes, wrong treatment, etc., and achieve the effect of simple operation

Inactive Publication Date: 2009-12-09
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • 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 vancomycin-resistant phenotype (Efm-HS0661, deposit number: CGMCC No. 2370) was obtained by conventional identification and drug susceptibility methods, and the universal primer for vancomycin-resistant gene vanF: 5'-GGGCTGCGATATTCAAAGCTC was used -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) of the drug resistance gene detection chip, and did not find the existence of the two genes; the nucleotide sequence ( figure 2 ), further used TaKaRa GenomeWalking Kit (Dalian Bao Bio Co., Ltd.) to carry out sequence sequencing and bioinformatics analysis of the two wings of the PCR product, and obtained a complete gene sequence of SEQ ID. The analysis software was compared, and the h...

Embodiment 2

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

[0037] The TaqMan probe 5'FAM-TTCGG CGGTTTGTATGGACAAATCAC-3'TAMERA was designed according to a unique sequence between the universal primers NvanF and NvanR in the above n...

Embodiment 3

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

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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 for mediating vancomycin resistance, in particular to a DNA sequence of a new gene that causes Enterococcus faecium to be resistant to vancomycin and its use. Background technique [0002] Enterococcus (Enterococcus spp.) is a part of the normal flora of the human intestine and genitourinary tract. It can also be used as an opportunistic pathogen to cause infection of various organs and tissues of 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 relevant reports, Enterococcus infection has been increasing in recent years, and it has become one of the main pathogens of nosocomial infection, ranking third among the pathogens that cause bloodstream infections such as sepsis. Enterococ...

Claims

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Application Information

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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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