Development of a Luminex xTAG® assay for cost-effective multiplex detection of ß-lactamases in Gram-negative bacteria.
J Antimicrob Chemother
; 71(9): 2479-83, 2016 09.
Article
em En
| MEDLINE
| ID: mdl-27287233
ABSTRACT
OBJECTIVES:
The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted ß-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP).METHODS:
A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(®) platform. We designed 46 xTAG(®)-compatible probes targeting ß-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates.RESULTS:
The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained <5 per sample.CONCLUSIONS:
We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Beta-Lactamases
/
Técnicas Bacteriológicas
/
Técnicas de Genotipagem
/
Bactérias Gram-Negativas
Tipo de estudo:
Diagnostic_studies
/
Health_economic_evaluation
Limite:
Humans
Idioma:
En
Revista:
J Antimicrob Chemother
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Bélgica