Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

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. 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.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

Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of <em>Bacillus anthracis, Francisella tularensis,</em> and <em>Yersinia pestis</em>

<div><h3>Background</h3><p>The intentional release of <em>Bacillus anthracis</em> in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents <em>Bacillus anthracis, Francisella tularensis,</em> and <em>Yersinia pestis,</em> are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses.</p> <h3>Methodology/Principal Findings</h3><p>We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species.</p> <h3>Conclusions/Significance</h3><p>The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents <em>Bacillus anthracis, Francisella tularensis,</em> and <em>Yersinia pestis</em> and clear discrimination from closely-related species and several environmental background strains. The assays may be extended to detect a large number of pathogens, are applicable to the evaluation of both environmental and clinical samples, and have the potential to be applied in military, public health, and clinical diagnostic settings.</p> </div

Rapid Multi-Locus Sequence Typing Using Microfluidic

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

30-plex PCR profiles generated from select biothreat agents <i>Ba, Ft</i>, and <i>Yp</i>.

<p>Electropherogram obtained from 100 copies of <i>Ba</i> strain Sterne (A); from 1000 copies of <i>Ft</i> subsp. <i>tularensis</i> WY96 (B); and from 100 copies of <i>Yp bv. mediavalis</i> strain KIM10v (C). Note that 1000 copies of <i>Ft</i> DNA are required to detect two of the FAM-labeled targets (<i>acp</i>A and <i>spe</i>A) due to their high AT content resulting in reduced PCR efficiency.</p

10-plex PCR profiles generated from representative <i>Ba</i> and <i>Ba</i>NN strains.

<p>As input to PCR, 100 copies of template <i>Ba</i> strains Sterne, Ames, and BA1035; and 1000 copies of template <i>Ba</i>NN strains <i>Bc</i> E33L and <i>Bt</i> 97-27, were used. The FAM (blue), JOE (green), TMR (black) and ROX (red) labeled products are aligned to illustrate amplicon sizes from the set of five strains. As expected, all 10 loci were amplified in the 3 <i>Ba</i> strains; only 6 of 10 loci were observed in <i>Ba</i>NNs. The <i>bas</i>B and <i>pbp</i>1A amplicons allow discrimination between strains Ames, Sterne, and BA1035; <i>pbp</i>1A fragment length also distinguishes <i>Bc</i> E33L from <i>Bt</i> 97-27. X-axes show fragment size in base pairs and Y-axes relative fluorescence units (rfu).</p

Efficiency and sensitivity of the 30-plex panel with real-world air filter samples.

<p>Electropherograms obtained from Pentagon filter (A); BioWatch filter I (B); and BioWatch filter II (C). Resulting profiles on the left were from amplification of DNAs purified from air filter; profiles on the right were from air filter DNAs spiked with 100 copies of <i>Ba</i> Sterne. Background noise was detected from direct amplification of the three air filter samples, and the 10-plex <i>Ba</i>-signature was generated for all <i>Ba</i>-spiked air filter DNA samples. The observed nonspecific peak at 444 bp (highlighted in red) was readily distinguished from the 10 specific product peaks (1-pXO1_<i>lef,</i> 2-<i>cod</i>Y, 3-<i>spo</i>VT, 4-<i>hem</i>L, 5-pXO1_<i>ger</i>XB, 6-<i>ssp</i>F, 7-<i>pbp</i>1A, 8-<i>yih</i>Y, 9-BA0872, and 10-<i>bas</i>B).</p

hAFLP types and Sequence Types (STs) of <i>Ft</i> strains and <i>Ft</i> near neighbor species DNA (shown in bold) and by <i>in silico</i> analysis of WGS (shown in italics).

<p>Corresponding Genbank accession numbers are in brackets. FLT: Fragment Length Type; GT: sequence genotype of individual locus; Δ: strain is deficient of this locus; n: no PCR product or sequence detected.</p

hAFLP types and Sequence Types (STs) of <i>Yp</i> strains and <i>Yp</i> near neighbor species DNA (shown in bold) and by <i>in silico</i> analysis of WGS strains (shown in italics).

<p>Corresponding Genbank accession numbers are in brackets. FLT: Fragment Length Type; GT: sequence genotype of individual locus; Δ: strain is deficient of this locus; n: no PCR product and no sequence detected; t: strain FV-1 has 3 copies of pPCP-<i>pla</i> locus in whole genome data belonging to GT 1 and 4;</p>*<p>hAFLP Type and ST assigned despite missing data (marked with “#”) in the WGS data.</p

hAFLP types and Sequence Types (STs) of <i>Ba</i> strain and <i>Ba</i> near neighbor species DNA determined experimentally (shown in bold) and by <i>in silico</i> analysis of WGS strains (shown in italics).

<p>Corresponding Genbank accession numbers are in brackets. FLT Fragment Length Type; GT: sequence genotype of locus;</p>*<p>hAFLP Type and ST assigned despite missing WGS data;</p>#<p>deletion or physical gap in WGS data; Δ: strain deficient of pXO1 plasmid or locus; n: no PCR product or sequence detected.</p

A multiplexed microfluidic PCR assay for sensitive and specific point-of-care detection of Chlamydia trachomatis.

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes.Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively.This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC