Detection of<i>Francisella tularensis</i>within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

Emanuel, Peter A.; Bell, Ryan P.; Dang, Jessica L.; McClanahan, Rebecca; David, J. C.; Burgess, Robert J.; Thompson, Joseph; Collins, L A; Hadfield, Ted L.

doi:10.1128/jcm.41.2.689-693.2003

Cited by 61 publications

(52 citation statements)

References 23 publications

(18 reference statements)

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“…The peptide sequencing capability of tandem mass spectrometry in iMALDI provides higher molecular specificity than ELISA. The sensitivity of iMALDI is comparable to the most sensitive PCR method [26], but may not be as rapid when the digestion and incubation time is considered. However, the digestion time of iMALDI can be greatly reduced by using immobilized trypsin [57], and the incubation time can be reduced by using an antibody with high affinity.…”

Section: Quantitationmentioning

confidence: 93%

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An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

Jiang

Parker

Fuller

et al. 2007

Analytica Chimica Acta

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Francisella tularensis (F. tularensis) has been designated by the CDC as one of the ten organisms most likely to be engineered for bioterrorism. Symptoms of tularemia in humans are non-specific, thus making the disease difficult to diagnose. If not quickly diagnosed and treated, the disease has a high mortality rate -thus methods for early and specific diagnosis are of critical importance. This immunoaffinity MALDI MS/MS (iMALDI) assay provides unambiguous detection of F.tularensis peptides at attomole levels from peptide solutions, and at low CFU levels from bacteria. The addition of stable-labeled versions of the peptide as internal standards allows absolute quantitation of F. tularensis peptides with a linear dynamic range spanning two orders of magnitude. The ability of mass spectrometry to obtain amino acid sequence data on affinity-captured peptides provides absolute specificity and avoids "false positives" from the non-specific binding. The F. tularensis iMALDI assay has been applied to different samples, such as nasal swabs.This novel quantitative diagnostic F. tularensis iMALDI assay allows the safe, sensitive and specific detection of F. tularensis. The assay can be easily adapted to other target peptides and therefore has broad application potential in clinical diagnosis of other pathogens and diseases.

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Section: Quantitationmentioning

confidence: 93%

“…A hand-held PCR assay could detect F. tularensis in 3 hours, and achieved the analytical sensitivity of 100 bacteria mL -1 PBS or 10 3 -10 4 bacteria mL -1 serum [26]. However, PCR may give false positives from contamination with other DNA, which lowers its specificity.…”

Section: Introductionmentioning

confidence: 99%

An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

Jiang

Parker

Fuller

et al. 2007

Analytica Chimica Acta

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“…The detailed development of DNA-RPA and RT-RPA was described for the assays for F. tularensis and RVFV, respectively, elsewhere (27,28).…”

Section: Resultsmentioning

confidence: 99%

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. Syndromic panels for infectious and emerging infectious diseases have been suggested to be of value in point-of-care (POC) diagnostics for developing countries and for biodefense (1). Since the introduction of molecular diagnostics and in particular real-time PCR, ample proof of its sensitivity and specificity has been generated. Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2-4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5-7).In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8-12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets. Nonspecific intercalating fluorophores or fluorescent primers have been used for real-time detection in LAMP, SDA, HDA, and RCA, and specific detection probe formats have been developed for NASBA, RCA, HDA,.In isothermal and exponential RPA, the phage recombinase UvsX and its cofactor UvsY form a nucleoprotein complex with oligonucleotide primers to scan for homologous sequences in a DNA template. Recognition of a specific homologous sequence leads to the initiation of strand invasion of the com...

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“…One group investigated the use of a handheld thermocycler, compared with laboratory-based PCR platform and culture of tissue homogenates from infected mice [43]. Culture of these tissues proved to be more sensitive than both PCR platforms.…”

Section: Pcrmentioning

confidence: 99%

Tularemia: current diagnosis and treatment options

Hepburn

Simpson

2008

Expert Review of Anti-infective Therapy

115

117

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Tularemia is an infection caused by Francisella tularensis with a worldwide distribution and diverse clinical manifestations. Limitations in both culture and serologic testing have led to substantial research into new diagnostic techniques and their clinical application, with PCR testing as the best example. This review focuses on the utility of culture, PCR and serologic testing for tularemia. In addition, we also review the evidence to support different therapeutic options for tularemia, highlighting both the most effective supporting evidence for therapeutic recommendations as well as gaps in current knowledge. We conclude the article with suggestions regarding potential areas for future research.

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Detection ofFrancisella tularensiswithin Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

Cited by 61 publications

References 23 publications

An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Tularemia: current diagnosis and treatment options

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