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Christian Massire,
Daelynn R Buelow,
Sean X Zhang,
Robert Lovari,
Heather E Matthews,
Donna M Toleno,
Raymond R Ranken, Thomas A Hall,
David Metzgar,
Rangarajan Sampath,
Lawrence B Blyn,
David J Ecker,
Zhengming Gu,
Thomas J Walsh,
Randall T Hayden
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ABSTRACT: Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 moulds, 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of moulds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.
Journal of clinical microbiology 01/2013; · 4.16 Impact Factor
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David D Duncan,
Amy J Vogler,
Mark J Wolcott,
Feng Li,
Derek S Sarovich,
Dawn N Birdsell,
Lindsey M Watson, Thomas A Hall,
Rangarajan Sampath,
Roberta Housley,
Lawrence B Blyn,
Steven A Hofstadler,
David J Ecker,
Paul Keim,
David M Wagner,
Mark W Eshoo
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ABSTRACT: A PCR assay was developed to genotypically characterize F. tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis, and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay. Letters in Applied Microbiology © 2012 Ibis Biosciences.
Letters in Applied Microbiology 11/2012; · 1.62 Impact Factor
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ABSTRACT: Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.
Forensic science international. Genetics 03/2012; 6(5):594-606. · 2.42 Impact Factor
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Rangarajan Sampath,
Niveen Mulholland,
Lawrence B Blyn,
Christian Massire,
Chris A Whitehouse,
Nicole Waybright,
Courtney Harter,
Joseph Bogan,
Mary Sue Miranda,
David Smith, [......],
Heather Matthews,
Donna Toleno,
Roberta Housley,
David Duncan,
Feng Li,
Robin Warren,
Mark W Eshoo, Thomas A Hall,
Steven A Hofstadler,
David J Ecker
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ABSTRACT: Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
PLoS ONE 01/2012; 7(6):e36528. · 4.09 Impact Factor
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David J Ecker,
Rangarajan Sampath,
Haijing Li,
Christian Massire,
Heather E Matthews,
Donna Toleno, Thomas A Hall,
Lawrence B Blyn,
Mark W Eshoo,
Raymond Ranken,
Steven A Hofstadler,
Yi-Wei Tang
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ABSTRACT: Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.
Expert Review of Molecular Diagnostics 05/2010; 10(4):399-415. · 4.86 Impact Factor
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Chris A Whitehouse,
Carson Baldwin,
Rangarajan Sampath,
Lawrence B Blyn,
Rachael Melton,
Feng Li, Thomas A Hall,
Vanessa Harpin,
Heather Matthews,
Marina Tediashvili,
Ekaterina Jaiani,
Tamar Kokashvili,
Nino Janelidze,
Christopher Grim,
Rita R Colwell,
Anwar Huq
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ABSTRACT: The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.
Applied and environmental microbiology 03/2010; 76(6):1996-2001. · 3.69 Impact Factor
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Donna M Wolk,
Lawrence B Blyn, Thomas A Hall,
Rangarajan Sampath,
Raymond Ranken,
Cristina Ivy,
Rachael Melton,
Heather Matthews,
Neill White,
Feng Li,
Vanessa Harpin,
David J Ecker,
Brandi Limbago,
Linda K McDougal,
Vicki H Wysocki,
Mian Cai,
Karen C Carroll
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ABSTRACT: There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus. Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections.
Journal of clinical microbiology 09/2009; 47(10):3129-37. · 4.16 Impact Factor
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ABSTRACT: We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.
Analytical Chemistry 08/2009; 81(18):7515-26. · 5.86 Impact Factor
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Thomas A Hall,
Rangarajan Sampath,
Lawrence B Blyn,
Raymond Ranken,
Cristina Ivy,
Rachael Melton,
Heather Matthews,
Neill White,
Feng Li,
Vanessa Harpin,
David J Ecker,
Linda K McDougal,
Brandi Limbago,
Tracy Ross,
Donna M Wolk,
Vicki Wysocki,
Karen C Carroll
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ABSTRACT: We describe a high-throughput assay using PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to determine the genotypes of Staphylococcus aureus isolates. The primer sets used in the PCR/ESI-MS assay were designed to amplify the same genes analyzed in multilocus sequence typing (MLST). The method was used to identify the clonal complex and USA type of each isolate and is suitable for use in a clinical or public-health setting. The method was validated using a panel of diverse isolates from the Centers for Disease Control and Prevention that were previously characterized by MLST and pulsed-field gel electrophoresis (PFGE). Clinical isolates from two geographically distinct hospitals were characterized, and the clustering results were in agreement with those for repetitive-element PCR and PFGE. The PCR/ESI-MS method enables genotyping of over 180 samples of S. aureus per day in an automated fashion.
Journal of clinical microbiology 04/2009; 47(6):1733-41. · 4.16 Impact Factor
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Carson D Baldwin,
Gerald B Howe,
Ranga Sampath,
Larry B Blyn,
Heather Matthews,
Vanessa Harpin, Thomas A Hall,
Jared J Drader,
Steve A Hofstadler,
Mark W Eshoo,
Karl Rudnick,
Karen Studarus,
David Moore,
Sharon Abbott,
J Michael Janda,
Chris A Whitehouse
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ABSTRACT: Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.
Diagnostic microbiology and infectious disease 03/2009; 63(4):403-8. · 2.45 Impact Factor
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ABSTRACT: We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
Methods in molecular biology (Clifton, N.J.) 02/2009; 551:71-87.
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Mark W Eshoo,
Chris A Whitehouse,
Aysegul Nalca,
Scott Zoll,
Joseph A Ecker, Thomas A Hall,
Thuy-Trang D Pennella,
David D Duncan,
Anjali Desai,
Emily K Moradi,
Karl Rudnick,
Brian Libby,
Raymond Ranken,
Rangarajan Sampath,
Steven A Hofstadler,
David J Ecker,
Lawrence B Blyn
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ABSTRACT: The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
PLoS ONE 02/2009; 4(7):e6342. · 4.09 Impact Factor
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ABSTRACT: We describe a new technology, the Ibis T5000, for the identification of pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis. The system enables the identification and quantification of a broad set of pathogens, including all known bacteria, all major groups of pathogenic fungi and the major families of viruses that cause disease in humans and animals, along with the detection of virulence factors and antibiotic resistance markers.
Nature Reviews Microbiology 07/2008; 6(7):553-8. · 21.18 Impact Factor
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James C Hannis,
Sheri M Manalili, Thomas A Hall,
Raymond Ranken,
Neill White,
Rangarajan Sampath,
Lawrence B Blyn,
David J Ecker,
Robert E Mandrell,
Clifton K Fagerquist,
Anna H Bates,
William G Miller,
Steven A Hofstadler
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ABSTRACT: In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.
Journal of clinical microbiology 04/2008; 46(4):1220-5. · 4.16 Impact Factor
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Lawrence B Blyn, Thomas A Hall,
Brian Libby,
Raymond Ranken,
Rangarajan Sampath,
Karl Rudnick,
Emily Moradi,
Anjali Desai,
David Metzgar,
Kevin L Russell,
Nikki E Freed,
Melinda Balansay,
Michael P Broderick,
Miguel A Osuna,
Steven A Hofstadler,
David J Ecker
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ABSTRACT: We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.
Journal of clinical microbiology 03/2008; 46(2):644-51. · 4.16 Impact Factor
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Mark W Eshoo,
Chris A Whitehouse,
Scott T Zoll,
Christian Massire,
Thuy-Trang D Pennella,
Lawrence B Blyn,
Rangarajan Sampath, Thomas A Hall,
Joseph A Ecker,
Anjali Desai,
Leonard P Wasieloski,
Feng Li,
Michael J Turell,
Amy Schink,
Karl Rudnick,
Glen Otero,
Scott C Weaver,
George V Ludwig,
Steven A Hofstadler,
David J Ecker
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ABSTRACT: Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.
Virology 12/2007; 368(2):286-95. · 3.35 Impact Factor
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ABSTRACT: Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety. Here we describe a powerful new approach for infectious disease surveillance that is based on polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry (ESI-MS) for accurate mass measurements of the PCR products, and base composition signature analysis to identify organisms in a sample. This approach is capable of automated analysis of more than 1,500 PCR reactions a day. It is applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens and will facilitate rapid characterization of known and emerging pathogens.
Annals of the New York Academy of Sciences 04/2007; 1102(1):109 - 120. · 3.15 Impact Factor
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ABSTRACT: Mitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants.
In a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis.
From the 24-sample blinded panel, we correctly identified 12 of the samples as bearing an mtDNA variant and found the remaining 12 samples to have no pathogenic variants. The correlation coefficient between the 2 methods for mtDNA variant detection was 1.0; there were no false positives or false negatives in this sample set. In addition, the ESI-FTICR method identified 4 single-nucleotide polymorphisms (SNP) that had previously been missed by standard PCR-RFLP analysis.
ESI-FTICR MS is a rapid, sensitive, and accurate method for the identification and quantification of mtDNA mutations and SNPs.
Clinical Chemistry 03/2007; 53(2):195-203. · 7.91 Impact Factor
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Rangarajan Sampath,
Kevin L Russell,
Christian Massire,
Mark W Eshoo,
Vanessa Harpin,
Lawrence B Blyn,
Rachael Melton,
Cristina Ivy,
Thuy Pennella,
Feng Li, [......],
Ginger Goekjian,
Samuel Yingst,
Marshall Monteville,
Magdi D Saad,
Chris A Whitehouse,
Carson Baldwin,
Karl H Rudnick,
Steven A Hofstadler,
Stanley M Lemon,
David J Ecker
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ABSTRACT: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.
Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.
Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.
PLoS ONE 01/2007; 2(5):e489. · 4.09 Impact Factor
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Joseph A Ecker,
Christian Massire, Thomas A Hall,
Raymond Ranken,
Thuy-Trang D Pennella,
Cristina Agasino Ivy,
Lawrence B Blyn,
Steven A Hofstadler,
Timothy P Endy,
Paul T Scott, [......],
Gregory Deye,
Scott Riddell,
Eric Milstrey,
Bruno Petruccelli,
Sylvain Brisse,
Vanessa Harpin,
Amy Schink,
David J Ecker,
Rangarajan Sampath,
Mark W Eshoo
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ABSTRACT: Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.
Journal of Clinical Microbiology 09/2006; 44(8):2921-32. · 4.15 Impact Factor
