Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips
Abstract
Multiple locus sequence typing (MLST) has become a central genotyping strategy for analysis of bacterial populations. The scheme involves de novo sequencing of 6-8 housekeeping loci to assign unique sequence types. In this work we adapted MLST to a rapid microfluidics platform in order to enhance speed and reduce laboratory labor time. Methodology/Principal Findings: Using two integrated microfluidic devices, DNA was purified from 100 Bacillus cereus soil isolates, used as a template for multiplex amplification of 7 loci and sequenced on forward and reverse strands. The time on instrument from loading genomic DNA to generation of electropherograms was only 1.5 hours. We obtained full-length sequence of all seven MLST alleles from 84 representing 46 different Sequence Types. At least one allele could be sequenced from a further 15 strains. The nucleotide diversity of B. cereus isolated in this study from one location in Rockville Maryland (0.04 substitutions per site) was found to be as great as the global collection of isolates. Conclusions/Significance: Biogeographical investigation of pathogens is only one of a panoply of possible applications of microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of human clinical samples.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 12, 2010
- Accession Number
- ADA552866
Entities
People
- Catherine C. Hogan
- Christopher Cook
- Eugen Tan
- Heidi Giese
- Richard F Selden
- Rosemary S. Turingan
- Timothy D Read
- Ulrich H. Thomann
Organizations
- Naval Medical Research Center