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

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

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

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

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

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

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

Comparison of multiplex and commercial Amplicor NAAT assays.

<p>Prior to discrepant resolution, 129 (49.0%) of 263 of the samples tested were detected positive by the multiplex PCR assay and 94 (35.7%) by Amplicor.</p

Sample population distributions of concordant and discordant samples (A) and results of multiplex PCR and Amplicor assays versus sequencing to resolve discordant calls (B).

<p>Numbers noted on each bar represent the number of samples that were or were not confirmed by sequencing. Following resolution of discrepants by sequencing, 129 (76+53) and 88 (76+12) samples were positive by multiplex and Amplicor assays, respectively. An overall concordance rate of 82.9% was found between the multiplex assay and sequencing as compared to 17.1% for the Amplicor assay.</p

Representative clinical sample 9-plex profiles.

<p>Approximately half the positive electropherograms showed all 9 amplicons with saturated (Panel A) or intermediate (Panel B) peak signals, suggesting high copy numbers of <i>Ct</i>. The remaining positive samples generated incomplete profiles, having fewer than 9 amplicons and suggesting low <i>Ct</i> numbers. For example, Panel C shows the pCT peak and three other amplicons, Panel D shows pCT and two other amplicons, and Panel E shows no pCT and two other amplicons in blue and red. Panel F is a negative clinical sample showing nonspecific peaks (circled in green) that are readily distinguished from <i>Ct</i> amplicons based on molecular weight and fluorescent dye color. The 353 bp peak in blue is readily distinguished from a true 349 bp pCT amplicon in blue. The 230 bp peak in red is readily distinguished from the 4 red-labeled specific amplicons, all of which differ in size from the artifact by at least 120 bp.</p