Lorelei Westin

Anchored multiplex amplification on a microelectronic chip array

We have developed a method for anchored amplification on a microchip array that allows amplification and detection of multiple targets in an open format. Electronic anchoring of sets of amplification primers in distinct areas on the microchip permitted primer-primer interactions to be reduced and distinct zones of amplification created, thereby increasing the efficiency of the multiplex amplification reactions. We found strand displacement amplification (SDA) to be ideal for use in our microelectronic chip system because of the isothermal nature of the assay, which provides a rapid amplification system readily compatible with simple instrumentation. Anchored SDA supported multiplex DNA or RNA amplification without decreases in amplification efficiency. This microelectronic chip-based amplification system allows multiplexed amplification and detection to be performed on the same platform, streamlining development of any nucleic acid-based assay.

Antimicrobial Resistance and Bacterial Identification Utilizing a Microelectronic Chip Array

ABSTRACT 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 Chlamydiacryptic 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.