Multiplex asymmetric PCR-based oligonucleotide microarray for detection of drug resistance genes containing single mutations in Enterobacteriaceae
Multiplex asymmetric PCR-based oligonucleotide microarray for detection of drug resistance genes containing single mutations in Enterobacteriaceae
复制标题
DOI：
10.1128/aac.01461-06
复制
发表时间：
2007-10-01
影响因子：
4.9
通讯作者：
Cheng, Jing
中科院分区：
文献类型：
--
作者：
Zhu, Ling-Xiang;Zhang, Zhi-Wei;Cheng, Jing
建立了一种基于多重不对称PCR（MAPCR）的基因芯片方法，用于检测革兰阴性菌中10种已知的超广谱β-内酰胺酶（ESBLs）和质粒介导的AmpC β-内酰胺酶基因，并对SHV基因中6个重要的点突变（35、43、130、179、238和240位氨基酸）进行分型。MAPCR基于两轮反应，通过采用多个通用的不相关序列标记引物并在第二轮扩增时提高退火温度来促进适于微阵列杂交的单链扩增子的积累。通过在等位基因特异性寡核苷酸探针中引入人工错配来提高微阵列的识别效率。微阵列检测正确地鉴定了参考菌株和111株临床分离株中的耐药基因，这些结果也通过直接DNA序列分析对一些分离株进行了证实。基因芯片检测的耐药基因型与表型MIC药敏试验密切相关。这种快速的MAPCR为基础的微阵列方法应证明是有用的进行重要的流行病学研究有关ESBLs和质粒介导的AmpC酶，也可以证明是宝贵的初步筛选，以补充临床诊断的表型检测。
A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC P-lactamase genes in gram-negative bacteria and for the typing of six important point mutations (amino acid positions 35, 43, 130, 179, 238, and 240) in the blas(SHV) gene. The MAPCR is based on a two-round reaction to promote the accumulation of the single-stranded amplicons amenable for microarray hybridization by employing multiple universal unrelated sequence-tagged primers and elevating the annealing temperature at the second round of amplification. A strategy to improve the discrimination efficiency of the microarray was constituted by introducing an artificial mismatch into some of the allele-specific oligonucleotide probes. The microarray assay correctly identified the resistance genes in both the reference strains and some 111 clinical isolates, and these results were also confirmed for some isolates by direct DNA sequence analysis. The resistance genotypes determined by the microarray correlated closely with phenotypic MIC susceptibility testing. This fast MAPCR-based microarray method should prove useful for undertaking important epidemiological studies concerning ESBLs and plasmid-mediated AmpC enzymes and could also prove invaluable as a preliminary screen to supplement phenotypic testing for clinical diagnostics.