Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance

Grimm, Verena; Ezaki, Satoshi; Šuša, Milorad; Knabbe, Cornelius; Schmid, Rolf D.; Bachmann, Till T.

doi:10.1128/jcm.42.10.4918.2004

Cited by 30 publications

(39 citation statements)

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“…One such application is the genotyping of SHV ␤-lactamases that confer resistance to extendedspectrum cephalosporins. Of the numerous post-PCR methods devised in the past, the most widely used are hybridizationbased assays including fluorescence-labeled oligonucleotide probes on a LightCycler instrument (25) or DNA microarrays (12), as well as PCR restriction analysis (2,18,23) and automated DNA sequencing (4). In this paper, we have introduced an improved method for comparative sequence analysis via base-specific cleavage of in vitro-transcribed RNA (29).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, a number of competing post-PCR techniques for fast identification have been developed during the past few years. For example, PCR-restriction fragment length polymorphism analysis (2,18,23), fluorescence-labeled oligonucleotide probes used on a LightCycler instrument (25), and DNA microarrays (12) have been proposed to identify some of the relevant point mutations. However, the major drawback of all of the current assays is that they are not able to identify previously unknown sequence variations.…”

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confidence: 99%

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Detection and Genotyping of SHV β-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Stürenburg

Storm²,

Sobottka

et al. 2006

J Clin Microbiol

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Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed bla SHV genes was used for the identification and genotyping of SHV ␤-lactamases. For evaluation, bla SHV stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Detection and Genotyping of SHV β-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Stürenburg

Storm²,

Sobottka

et al. 2006

J Clin Microbiol

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“…In view of the increasing number of fungal species isolated from clinical samples as potential pathogens, a broad-spectrum detection system as it is represented by the microarray technology would be desirable. Until today, diagnostic DNA microarrays were applied for the identification of viruses (5,(27)(28)(29)56), bacteria (14,24,45,46,49), and mechanisms of resistance to certain antibiotics (15,18,55). To our knowledge, this study describes for the first time the application of the DNA microarray technology in combination with a quantification and data-processing method for the rapid and reliable detection of fungal pathogens.…”

Section: Discussionmentioning

confidence: 99%

Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses

et al. 2005

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Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is timeconsuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS1 and ITS4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 precharacterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.

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“…DNA microarrays are used in three major clinical areas: (a) for gene expression profiling -measuring the expression level of thousands of genes in any tissue sample, (b) for genotyping -determination of diseaserelevant genes or agents causing diseases, and (c) DNA sequencing -screening thousands of DNA base pairs for mutations in specific genes whose normal sequence is already known (screening of single nucleotide polymorphisms, SNPs) 57 . Grimm et al 58 developed and validated oligonucleotide microarray for the rapid identification of ESBL in Gram-negative bacteria by simultaneously genotyping bla TEM , bla SHV and bla CTX-M . The array consisted of 168 probes which cover mutations responsible for 156 amino acid substitutions with the assay time of 5 hours.…”

Section: Real-time Pcrmentioning

confidence: 99%

GENETIC METHODS FOR DETECTION OF ANTIBIOTIC RESISTANCE: FOCUS ON EXTENDED-SPECTRUM Β-Lactamases

Chromá¹,

Kolář²

2010

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

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Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance

Cited by 30 publications

References 0 publications

Detection and Genotyping of SHV β-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Detection and Genotyping of SHV β-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses

GENETIC METHODS FOR DETECTION OF ANTIBIOTIC RESISTANCE: FOCUS ON EXTENDED-SPECTRUM Β-Lactamases

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