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

Turingan, Rosemary S.; Thomann, Hans-Ulrich; Zolotova, Anna; Tan, Eugene; Selden, Richard F.

doi:10.1371/journal.pone.0056093

Cited by 14 publications

(12 citation statements)

References 55 publications

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“…Targeted amplification of organism-specific regions prior to sequencing has shown some promise. For instance, an assay in which multiplex PCR preceded sequencing was able to fully differentiate Bacillus anthracis, Yersinia pestis and Francisella tularensis [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

et al. 2014

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BackgroundThe introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown.ResultsIn the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis.ConclusionThe analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.

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Section: Discussionmentioning

confidence: 99%

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

et al. 2014

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“…Select PCR amplicons were subjected to microfluidic Sanger sequencing as previously described [ 34 ]. PCR products were directly used as template for microfluidic sequencing reactions using the BigDye ® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA).…”

Section: Methodsmentioning

confidence: 99%

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay

Turingan¹,

Kaplun²,

Krautz‐Peterson³

et al. 2017

PLoS ONE

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Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.

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“…We will discuss the detail development of PCR below as exemplified by a recent publication [ 23 ] and focus on the findings about F . tularensis .…”

Section: Identification and Characterization Of Isolated Frmentioning

confidence: 99%

Rapid Identification and Characterization of Francisella by Molecular Biology and Other Techniques

Lai¹,

Zhao²,

Chen³

et al. 2016

TOMICROJ

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Francisella tularensis is the causative pathogen of tularemia and a Tier 1 bioterror agent on the CDC list. Considering the fact that some subpopulation of the F. tularensis strains is more virulent, more significantly associated with mortality, and therefore poses more threat to humans, rapid identification and characterization of this subpopulation strains is of invaluable importance. This review summarizes the up-to-date developments of assays for mainly detecting and characterizing F. tularensis and a touch of caveats of some of the assays.

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

Cited by 14 publications

References 55 publications

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay

Rapid Identification and Characterization of Francisella by Molecular Biology and Other Techniques

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