Multiplex asymmetric PCR-based oligonucleotide microarray for detection of drug resistance genes containing single mutations in Enterobacteriaceae.
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 beta-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 bla(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.
Zhu
Ling-Xiang
LX
Medical Systems Biology Research Center, Tsinghua University School of Medicine, Beijing, PR China.
Zhang
Zhi-Wei
ZW
Liang
Dong
D
Jiang
Di
D
Wang
Can
C
Du
Ning
N
Zhang
Qiong
Q
Mitchelson
Keith
K
Cheng
Jing
J
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002681:China; D017931:DNA Primers; D003627:Data Interpretation, Statistical; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004755:Enterobacteriaceae / Q000235:genetics* / Q000502:physiology; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; C048795:AmpC beta-lactamases; D001618:beta-Lactamases