rapid multi-locus sequence typing using microfluidic biochips快速多位点序列输入使用微流控生物芯片.pdfVIP

Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips 1,2 3 1 3 3 Timothy D. Read *, Rosemary S. Turingan , Christopher Cook , Heidi Giese , Ulrich Hans Thomann , 3 3 3 Catherine C. Hogan , Eugene Tan , Richard F. Selden 1 Biological Defense Research Directorate, Naval Medical Research Center, Rockville, Maryland, United States of America, 2 Division of Infectious Diseases, Department of Medicine, and Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America, 3 Network Biosystems Inc, Woburn, Massachusetts, United States of America Abstract 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. 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 fo