Biological Defense Research Directorate, Naval Medical Research Center, Rockville, Maryland, United States of America.
PLoS One. 2010 May 12;5(5):e10595. doi: 10.1371/journal.pone.0010595.
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.
多位点序列分型（MLST）已成为分析细菌种群的核心基因分型策略。该方案涉及从头测序 6-8 个管家基因座以分配独特的序列类型。在这项工作中，我们将 MLST 改编到快速微流控平台上，以提高速度并减少实验室劳动时间。
方法/主要发现：使用两个集成的微流控设备，从 100 个蜡状芽孢杆菌土壤分离物中提取 DNA，用作 7 个基因座的多重扩增模板，并在正向和反向链上测序。从加载基因组 DNA 到生成电泳图谱的仪器时间仅为 1.5 小时。我们从代表 46 种不同序列类型的 84 个中获得了所有 7 个 MLST 等位基因的全长序列。另外 15 株至少可以测序一个等位基因。在马里兰州罗克维尔的一个地点从本研究中分离的蜡状芽孢杆菌的核苷酸多样性（每个位点 0.04 个取代）与全球分离物一样大。
结论/意义：基于微流控的 MLST 的生物地理学研究仅是病原体的多种可能应用之一；其他应用包括微生物取证、生物威胁识别和人类临床样本的快速特征描述。
