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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 t...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868872/ https://www.ncbi.nlm.nih.gov/pubmed/20485679 http://dx.doi.org/10.1371/journal.pone.0010595 |
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author | Read, Timothy D. Turingan, Rosemary S. Cook, Christopher Giese, Heidi Thomann, Ulrich Hans Hogan, Catherine C. Tan, Eugene Selden, Richard F. |
author_facet | Read, Timothy D. Turingan, Rosemary S. Cook, Christopher Giese, Heidi Thomann, Ulrich Hans Hogan, Catherine C. Tan, Eugene Selden, Richard F. |
author_sort | Read, Timothy D. |
collection | PubMed |
description | 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 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. |
format | Text |
id | pubmed-2868872 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-28688722010-05-19 Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips Read, Timothy D. Turingan, Rosemary S. Cook, Christopher Giese, Heidi Thomann, Ulrich Hans Hogan, Catherine C. Tan, Eugene Selden, Richard F. PLoS One Research Article 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 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. Public Library of Science 2010-05-12 /pmc/articles/PMC2868872/ /pubmed/20485679 http://dx.doi.org/10.1371/journal.pone.0010595 Text en Read et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Read, Timothy D. Turingan, Rosemary S. Cook, Christopher Giese, Heidi Thomann, Ulrich Hans Hogan, Catherine C. Tan, Eugene Selden, Richard F. Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title | Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title_full | Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title_fullStr | Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title_full_unstemmed | Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title_short | Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips |
title_sort | rapid multi-locus sequence typing using microfluidic biochips |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868872/ https://www.ncbi.nlm.nih.gov/pubmed/20485679 http://dx.doi.org/10.1371/journal.pone.0010595 |
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