Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae
Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae
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DOI：
10.1093/jac/dkp498
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发表时间：
2010-03-01
影响因子：
5.2
通讯作者：
Arlet, Guillaume
中科院分区：
文献类型：
--
作者：
Dallenne, Caroline;Da Costa, Anaelle;Arlet, Guillaume
开发一种快速可靠的工具，通过多重PCR检测最常见的编码OXA-1样广谱β-内酰胺酶、超广谱β-内酰胺酶（ESBLs）、质粒介导的AmpC β-内酰胺酶和A、B和D类碳青霉烯酶的β-内酰胺酶基因。建立了一组六个多重PCR和一个单一PCR。使用从临床标本中分离的31株肠杆菌科菌株进行了评价，这些菌株对广谱头孢菌素和/或头孢霉素和/或碳青霉烯类具有耐药表型。PCR产物直接测序鉴定β-内酰胺酶基因，在优化条件下，所有阳性对照均证实了组特异性PCR引物的特异性。除了检测碳青霉烯酶基因外，使用相同的PCR条件进行多重和单纯PCR试验，允许在单次运行中进行试验。31株分离株中，22株产ESBL，主要为CTX-M-15，但也有CTX-M-1、CTX-M-9、SHV-12、SHV-5、SHV-2、TEM-21、TEM-52和1株VEB型ESBL，6株产质粒介导的AmpC酶（5株DHA-1和1株CMY-2）和3株产ESBL（两架SHV-12，一种CTX-M-15）和一种质粒介导的AmpC β-内酰胺酶（DHA-1）本文报道了一种由六种多重PCR和一种单重PCR组成的快速筛选常见β-内酰胺酶的方法。该方法允许从PCR产物直接测序。
To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases.Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes.Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1).We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.