Oligonucleotide microarrays with horseradish peroxidase-based detection for the identification of extended-spectrum β-lactamases
Oligonucleotide microarrays with horseradish peroxidase-based detection for the identification of extended-spectrum β-lactamases
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DOI：
10.1016/j.bios.2010.06.053
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发表时间：
2010-12-15
影响因子：
12.6
通讯作者：
Egorov, Alexey M.
中科院分区：
文献类型：
--
作者：
Rubtsova, Mayya Yu.;Ulyashova, Maria M.;Egorov, Alexey M.
产超广谱β-内酰胺酶（ESBLs）是肠杆菌科细菌对现代β-内酰胺类抗生素产生耐药的重要机制之一。已知有400种ESBLs，其中大部分由TEM、SHV和CTX-M家族的酶代表。利用辣根过氧化物酶（HRP）建立了检测ESBLs和耐药β-内酰胺酶的寡核苷酸芯片。已经设计了特异性寡核苷酸探针用于鉴定β-内酰胺酶家族和负责拓宽底物特异性和抑制剂耐受性的重要SNP。建立了同时扩增TEM、SHV和CTX-M型β-内酰胺酶全长基因并用生物素标记的多重PCR方法。然后将标记的靶DNA与固定在多孔膜支持物上的特异性寡核苷酸探针杂交。杂交后，生物素标记的DNA双链体用比色法检测的链霉亲和素-HRP偶联物染色。通过设计寡核苷酸探针和优化杂交条件，保证了所有对照ESBLs鉴定的特异性。新开发的方法已成功用于鉴定90株肠杆菌科临床分离株中的bla（TEM）、bla（SHV）和bla（CTX-M）基因：发现70%携带bla（TEM）、50%携带bla（SHV）、50%携带bla（CTX-M）; CTX-M亚群分布如下：68%携带bla（CTX-M-1）、4%携带bla（CTX-M-2）、14%携带bla（CTX-M-9）。未检出TEM型和IRT型ESBLs，24.6%的临床标本同时检出TEM型和SHV型ESBLs。CTX-M-1样和SHV-5样基因的混合物是检测到的最丰富的组合。膜微阵列技术结合比色检测技术可提供高特异性和有效性的ESBL和IRT产生样品的筛选。(C)2010爱思唯尔有限公司版权所有。
Production of extended-spectrum beta-lactamases (ESBLs) is the one of most widespread and clinically significant mechanism of Enterobacteriaceae resistance towards modern beta-lactam antibiotics. There are known 400 ESBLs, with the majority represented by the enzymes of TEM, SHV and CTX-M families. Oligonucleotide microarrays with colorimetric detection have been developed for the purposes of determination of ESBLs and inhibitor-resistant beta-lactamases using horseradish peroxidase (HRP). Specific oligonucleotide probes have been designed for the identification of beta-lactamase family and important SNPs responsible for the broadening of substrate specificity and tolerance to inhibitors. Multiplex PCR has been developed for simultaneous amplification and labeling of full-size genes of TEM-, SHV- and CTX-M-type beta-lactamases with biotin. The labeled target DNA is then hybridized with specific oligonucleotide probes immobilized on a porous membrane support. After hybridization, biotin-labeled DNA duplexes are stained with the streptavidin-HRP conjugate detected colorimetrically. Design of oligonucleotide probes and optimization of hybridization conditions ensure the specificity of all control ESBLs identification. The newly developed method has been successfully used to identify bla(TEM), bla(SHV) and bla(CTX-M) genes in 90 clinical isolates of Enterobacteriaceae: 70% were found to carry bla(TEM), 50% bla(SHV), 50% bla(CTX-M); with the following distribution of CTX-M subclusters: 68% bla(CTX-M-1), 4% bla(CTX-M-2), and 14% bla(CTX-M-9). No ESBL of TEM-type and IRT phenotype assigned to TEM- or SHV-type beta-Iactamases had been detected; 24.6% of clinical samples show two types of ESBLs simultaneously. A mixture of CTX-M-1-like and SHV-5-like genes was the most abundant combination detected. Membrane microarray technique with colorimetric detection provides both high specificity and effectiveness of screening for ESBL- and IRT-producing samples. (C) 2010 Elsevier B.V. All rights reserved.