Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array.
Species-specific bacterial identification of clinical specimens is often limited to a few species due to the difficulty of performing multiplex reactions. In addition, discrimination of amplicons is time-consuming and laborious, consisting of gel electrophoresis, probe hybridization, or sequencing technology. In order to simplify the process of bacterial identification, we combined anchored in situ amplification on a microelectronic chip array with discrimination and detection on the same platform. Here, we describe the simultaneous amplification and discrimination of six gene sequences which are representative of different bacterial identification assays: Escherichia coli gyrA, Salmonella gyrA, Campylobacter gyrA, E. coli parC, Staphylococcus mecA, and Chlamydia cryptic plasmid. The assay can detect both plasmid and transposon genes and can also discriminate strains carrying antibiotic resistance single-nucleotide polymorphism mutations. Finally, the assay is similarly capable of discriminating between bacterial species through reporter-specific discrimination and allele-specific amplification. Anchored strand displacement amplification allows multiplex amplification and complex genotype discrimination on the same platform. This assay simplifies the bacterial identification process greatly, allowing molecular biology techniques to be performed with minimal processing of samples and practical experience.
Department of Advanced Research, Nanogen, Inc., 10398 Pacific Center Ct., San Diego, CA 92121, USA.
L
L
Westin
C
C
Miller
D
D
Vollmer
D
D
Canter
R
R
Radtkey
M
M
Nerenberg
J P
JP
O'Connell
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000145:classification* / Q000187:drug effects* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial; D005784:Gene Amplification; D008904:Miniaturization; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial