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Antimicrobial Agents and Chemotherapy, October 2007, p. 3707-3713, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.01461-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiplex Asymmetric PCR-Based Oligonucleotide Microarray for Detection of Drug Resistance Genes Containing Single Mutations in Enterobacteriaceae{triangledown}

Ling-Xiang Zhu,1,2,3 Zhi-Wei Zhang,3,4 Dong Liang,1,2,3 Di Jiang,3,4 Can Wang,3,4 Ning Du,3,4 Qiong Zhang,3,4 Keith Mitchelson,1,3,4 and Jing Cheng1,2,3,5*

Medical Systems Biology Research Center, Tsinghua University School of Medicine, Beijing 100084, People's Republic of China,1 Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China,2 National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Changping District, Beijing 102206, People's Republic of China,3 CapitalBio Corporation, 18 Life Science Parkway, Changping District, Beijing 102206, People's Republic of China,4 State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China5

Received 21 November 2006/ Returned for modification 21 March 2007/ Accepted 30 May 2007

A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-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 blaSHV 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.

* Corresponding author. Mailing address: Medical Systems Biology Research Center, Tsinghua University School of Medicine, Beijing 100084, People's Republic of China. Phone: 86-10-62772239. Fax: 86-10-80726898. E-mail: jcheng{at}tsinghua.edu.cn

{triangledown} Published ahead of print on 23 July 2007.

Antimicrobial Agents and Chemotherapy, October 2007, p. 3707-3713, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.01461-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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