Abstract
BackgroundMultiple 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 FindingsUsing 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/SignificanceBiogeographical 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.
Highlights
Multiple locus sequence typing, developed in the early 1990s [1], has become a central genotyping strategy for analysis of bacterial populations
The thermal cycler (Figure 1a) consisted of three main subsystems: a heat pump providing a variable temperature-controlled surface on which the microfluidic biochip (Figure 1b) is positioned, a compression case that clamps the biochip to the heat pump, and electronic hardware and software that accurately control and maintain cycled temperature profile
Electrophoretic separation of DNA fragments that were the product of the Sanger sequencing reactions took place within a second microfluidic biochip on the Genebench-FX instrument prefilled with sieving matrix designed and optimized for rapid, high resolution and high sensitivity separation performance
Summary
Multiple locus sequence typing, developed in the early 1990s [1], has become a central genotyping strategy for analysis of bacterial populations. For a typical scheme, conserved oligonucleotide primers are designed to amplify 300–600 bp fragments of 6–8 housekeeping genes. Bacterial strains are assigned to unique combinations of alleles called Sequence Types (STs). Whole genomic shotgun sequencing using ‘nextgeneration’ sequencing technologies [3] is orders of magnitude more efficient on a per-nucleotide basis, MLST is a more rapid and cost effective method for ascertaining the genetic structure of large strain collections, when many of the isolates may be isogenic. 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
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