Rapid multi-locus sequence typing using microfluidic biochips.
BACKGROUND
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.


RESULTS
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
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.
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Timothy D
TD
Biological Defense Research Directorate, Naval Medical Research Center, Rockville, Maryland, United States of America. tread@emory.edu
Turingan
Rosemary S
RS
Cook
Christopher
C
Giese
Heidi
H
Thomann
Ulrich Hans
UH
Hogan
Catherine C
CC
Tan
Eugene
E
Selden
Richard F
RF
D001409:Bacillus cereus / Q000145:classification / Q000235:genetics*; D015373:Bacterial Typing Techniques; D004783:Environmental Microbiology; D056426:Genetic Loci / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature