Rapid DNA analysis for automated processing and interpretation of low DNA content samples

Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time

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

Fully integrated, fully automated generation of short tandem repeat profiles

Background: The generation of short tandem repeat profiles, also referred to as ‘DNA typing,’ is not currently performed outside the laboratory because the process requires highly skilled technical operators and a controlled laboratory environment and infrastructure with several specialized instruments. The goal of this work was to develop a fully integrated system for the automated generation of short tandem repeat profiles from buccal swab samples, to improve forensic laboratory process flow as well as to enable short tandem repeat profile generation to be performed in police stations and in field-forward military, intelligence, and homeland security settings. Results: An integrated system was developed consisting of an injection-molded microfluidic BioChipSet cassette, a ruggedized instrument, and expert system software. For each of five buccal swabs, the system purifies DNA using guanidinium-based lysis and silica binding, amplifies 15 short tandem repeat loci and the amelogenin locus, electrophoretically separates the resulting amplicons, and generates a profile. No operator processing of the samples is required, and the time from swab insertion to profile generation is 84 minutes. All required reagents are contained within the BioChipSet cassette; these consist of a lyophilized polymerase chain reaction mix and liquids for purification and electrophoretic separation. Profiles obtained from fully automated runs demonstrate that the integrated system generates concordant short tandem repeat profiles. The system exhibits single-base resolution from 100 to greater than 500 bases, with inter-run precision with a standard deviation of ±0.05 - 0.10 bases for most alleles. The reagents are stable for at least 6 months at 22°C, and the instrument has been designed and tested to Military Standard 810F for shock and vibration ruggedization. A nontechnical user can operate the system within or outside the laboratory. Conclusions: The integrated system represents the first generation of a turnkey approach to short tandem repeat profiling and has the potential for use in both the field (for example, police booking stations, the battlefield, borders and ports) and the forensic laboratory

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