OS16 - 4.P4.c Tailed Primers Enhance Real-Time RT-PCR Detection of FMD Virus - D. Lefebvre
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This document summarizes a presentation on enhancing the specificity of real-time RT-PCR detection of foot-and-mouth disease virus through the use of tailed primers. The presentation evaluated adding tails to the primers for two different gene targets, 5'-UTR and 3D, and found that tailed primers improved detection of FMDV, with tailed 3D primers showing a consistent Cq value shift across multiple virus strains. Tailed primers enhance the robustness and performance of RT-PCR assays for FMDV detection and can help authorities demonstrate that a sample does not contain the virus by improving specificity and reducing risks of contamination, cross-reactivity, or detecting small amounts of viral RNA.
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current standard approach for detecting the UTI pathogens is culture based. This method is time consuming, has low throughput, and can lack sensitivity and/or specificity. In addition, not all uropathogens grow equally well under standard culture conditions which can result in a failure to detect the species. To address these gaps, we have developed a unique workflow from sample preparation to target identification using the nanofluidic OpenArray™ platform for spatial multiplexing of target specific assays. In this study, we tested pre-determined blinded research samples and confirmed the subset of results with orthogonal Sanger sequences.2011 pcr rflp to anopheles
This document describes the development of a PCR-RFLP assay to identify Plasmodium species and variants of P. vivax infecting Anopheles mosquitoes. Specific primers were designed that target regions of the circumsporozoite gene to distinguish P. falciparum, P. malariae, and P. vivax variants VK210, VK247, and P. vivax-like. The assay was tested on artificially infected mosquitoes and showed good agreement with nested PCR. The PCR-RFLP method provides a sensitive way to detect Plasmodium species and variants, which can help understand malaria transmission dynamics.
LSD symposium - A. Haegeman - LSDV transmission by stomoxys stable flies less...
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section 9 is highlighted- 3- Focus on Section 9- How can researchers.pdf
section 9 is highlighted.
3. Focus on Section 9. How can researchers and conservationists test for the presence of the
prion associated with CWD? Why is testing difficult? What is the treatment? Do your answers to
these questions make sense given your knowledge of protein structure and folding? for CWD
transmission to humans, one report rectal mucos, and brain in CWD positive elk. indicates
nonhuman primate susceptibility to alhough one of seven elk with early CWD CWD.
Intracerebral inoculation of squirrel was tonsil positive, but rectal mucosa negamonkeys (Saimini
sciureus) demonstrated a tive [82]. positive CWD transmission [49]. Among non- PrP detection
in CWD-infected deer human primates, however, the Pmp sequence: blood has been a challenge
to develop as an anof the new world monkeys are the most dis- temortem diagnostic tool. Prptic
was recently tant from humans [72], and therefore may not reported as detectable in deer blood
using an indicate that human prion protein conversion antibody ELISA with signal amplification
of would be induced by CWD PrP S c . antibody conjugated DNA catalyzed by T7 RNA
polymerase [15]. 1. CWD DIAGNOSTICS Protein misfolding cyclic amplification (PMCA) can
be used to amplify PrPir from Clinical kigns of CWD in deer and elk CWD-infected deer, and
should be valuable are unspecific and-subtle in cariy disease and for Prpes detection from tissues
and body flucommonly include weight loss and behav- ids containing low levels of infectivity.
for foral changes such as isolation from the herd improved understanding of CWD pathogeneand
depression. Other signs may include hy: improved understanding of CWD palso to detect
animals in early stages persalivation, polydipsia/polyuria, ataxia, and of infection [43]. Atarashi
et al. [3] developed occisionally increased regurgitation and/or of infection [43]. Atarashi ct al.
[3/ developed esophageal distension. Therefore sensitive, nique whereby recombinant, natively
folded specific, and rapid ante-mortem CWD ascays Pique whereby recombinant, natively folded
are eritical for accurafe diagnosis. Removal PrP monomers is used to amp 50 ag of PrPs was
detectable. Therefore of infected animals from a berd, pairticularly methods for sensitive
detection of Prpe are in US National Parks where culling of only subatantially improvingl known
infected individuals is often the preferred method of management ( M . Wild, personal
communication). To survey and man- 10. TOOLS FOR CWD RESEARCH: CELL. age CWD in
Rocky Mountain National Park IINES AND CERVID PrP EXPRESSING and adjacent Estes
Park in Colondo.male TRANSGENIC MICE deer were anesthetized, radiocollared, and ton-
Until recently, few techniques were availsil biopsies collected and tected (M. Wild, able to detect
CWD prion infectivity, or to personal communication: [941) Any prion: test compounds to
disrupt prion conversion. A infected animals were then located by ra- CWD-susceptible cell line
derived from cervid diotelemetry and euthani.
es062912s
This document describes a study that developed and validated a multiplex real-time PCR assay to distinguish between human, bovine, and swine fecal contamination in water samples. Species-specific primers and probes were created to target the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of each species. The assay was able to correctly identify the species in spiked effluent samples 83% of the time with no false positives. Some carry-over mitochondrial DNA signal was detected in human feces after consuming beef but not pork products. The multiplex real-time PCR provides a promising new tool for identifying the source of fecal contamination in environmental samples.
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26 - 28 /10/2016
EuFMD Sessions\Open Session\Archive-2018\Open 2016 Cascais- Portugal\PPT presentations\
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This study aimed to standardize PCR-based systems for the diagnosis of lymphatic filariasis using serum and urine samples. Paired biological samples were collected from 20 individuals known to be infected with Wuchereria bancrofti. Conventional and semi-nested PCR assays were optimized to detect W. bancrofti DNA. The internal PCR system was able to detect as little as 10 fg of W. bancrofti DNA and detected infection in all 20 patients using both serum and urine samples. In contrast, the semi-nested PCR only detected infection in 2 of the 20 patients. This study demonstrates the potential of using a simple internal PCR system and urine samples for the diagnosis of W. b
Tuberculosis diagnostics
Information about diagnostic methods and techniques for tuberculosis including microscopy, fluorescence microscopy, mycobacterial culture, molecular techniques (line probe assay, Xpert MTB/RIF), interferon gamma release assay (IGRA) and tuberculin skin test (TST)
Poster benito SIDiLV BVD/BDV
The one-step RT-qPCR assay developed by Exopol S.L. was able to detect a wide variety of ruminant pestiviruses with excellent specificity and good sensitivity down to 102 copies per reaction. Testing on 272 clinical samples identified BVDV/BDV in 17.1% of cattle and 16.7% of sheep samples, with most positive cases in reproductive swabs and blood/serum. The assay provides a reliable and sensitive diagnostic tool for routine detection of pestiviruses in ruminants.
Amany m. elshamy why the negative covid19 pcr test is a misguide results
The document discusses limitations of PCR tests for COVID-19 diagnosis. PCR tests have sensitivity of 70-90% and false negatives are common within the first week of infection or after 15 days. Commercial PCR kits target genes like ORF1ab, E, N and RdRP which have high genetic diversity and mutations, reducing accuracy. Serology tests have increasing sensitivity after the first week but depend on viral activity and cannot determine active infection. Chest CT is more reliable than PCR for diagnosis, showing characteristic lung opacities. Improved COVID-19 diagnostics are needed to enhance early reporting and accurately diagnose asymptomatic cases.
Sars Varna Sakratena
This document summarizes key details about the 2003 SARS outbreak:
1) The outbreak originated in Guangdong Province, China in November 2002 and eventually spread to over two dozen countries, infecting over 8,000 people and killing nearly 800.
2) The causative agent was identified as a novel coronavirus (SARS-CoV) through collaborative research between 11 laboratories in 9 countries.
3) SARS transmission occurred primarily in healthcare settings and through super-spreader events, with the earliest cases linked to food markets exposing humans to civets and racoon dogs carrying the virus.
2007 pcrrflp csp variants
1. The study developed a new PCR/RFLP technique to identify the 3 genotypes of Plasmodium vivax circumsporozoite protein (VK210, VK247, and P. vivax-like) using DNA extracted from blood samples.
2. The technique uses PCR amplification of the central immunodominant region of the CSP gene followed by restriction enzyme digestion and fragment analysis to distinguish the genotypes.
3. Testing demonstrated the technique could accurately identify the genotypes using plasmid controls for each variant, and that it had high sensitivity detecting parasitemia levels as low as 0.0069 parasites per microliter.
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VADEMOS is a decision support tool created by the European Commission for the Control of Foot-and-Mouth Disease to estimate current and future vaccine demand for foot-and-mouth disease at national and regional levels. It uses factors like livestock population forecasts, disease control policies, vaccination schedules, and outbreak forecasts with data from sources like WOAH and FAOSTAT. The model provides outputs on expected vaccine doses needed by geography, type of vaccination, species, and year over a 10-year period. While validation is needed, the tool generally predicts vaccine needs within calculated ranges, though estimates are sometimes too high. Future work will refine inputs, add additional geographical specificity, and expand the model to other diseases.
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bordetella pertussis.................................ppt
The binding of cosmological structures by massless topological defects
The binding of cosmological structures by massless topological defects
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...
OS16 - 4.P4.c Tailed Primers Enhance Real-Time RT-PCR Detection of FMD Virus - D. Lefebvre
- 1. Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016 TAILED PRIMERS ENHANCE REAL- TIME RT-PCR DETECTION OF FOOT AND MOUTH DISEASE VIRUS F. Vandenbussche, E. Mathijs, D. Lefebvre, K. De Clercq and S. Van Borm
- 2. Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus. Callahan JD, Brown F, Osorio FA, Sur JH, Kramer E, Long GW, Lubroth J, Ellis SJ, Shoulars KS, Gaffney KL, Rock DL, Nelson WM. J Am Vet Med Assoc. 2002 Jun 1;220(11):1636-42. 3D 5’-UTR 3D and 5’-UTR Reference methods
- 3. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV…
- 4. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV… • Contamination ? • Cross-reactivity ? • PCR artefact ? • … • Small amount of FMD viral RNA ?
- 5. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV… • Contamination ? • Cross-reactivity ? • PCR artefact ? • … • Small amount of FMD viral RNA ? To improve the robustness and overall performance of the assays ?!
- 7. 5’-UTR: dilution series with tailed and non-tailed primers
- 8. 5’-UTR: effect of tailing of forward and/or reverse primers
- 9. 3D: 5 / 50 FMDV strains showed Cq value shift (3.43 ± 0.11)
- 10. 3D: 5 / 50 FMDV strains showed Cq value shift (3.43 ± 0.11)
- 12. Thank you for your attention!