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Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

Authors
Publisher
Biomed Central Ltd
Publication Date
Keywords
  • Background: Ovine Footrot Is A Contagious Disease With Worldwide Occurrence In Sheep
  • The Main Causative Agent Is The Fastidious Bacterium Dichelobacter Nodosus
  • In Scandinavia
  • Footrot Was First Diagnosed In Sweden In 2004 And Later Also In Norway And Denmark
  • Clinical Examination Of Sheep Feet Is Fundamental To Diagnosis Of Footrot
  • But D
  • Nodosus Should Also Be Detected To Confirm The Diagnosis
  • Pcr-Based Detection Using Conventional Pcr Has Been Used At Our Institutes
  • But The Method Was Laborious And There Was A Need For A Faster
  • Easier-To-Interpret Method
  • The Aim Of This Study Was To Develop A Taqman-Based Real-Time Pcr Assay For Detection Of D
  • Nodosus And To Compare Its Performance With Culturing And Conventional Pcr
  • Methods: A D
  • Nodosus-Specific Taqman Based Real-Time Pcr Assay Targeting The 16S Rrna Gene Was Designed
  • The Inclusivity And Exclusivity (Specificity) Of The Assay Was Tested Using 55 Bacterial And Two Fun
  • To Evaluate The Sensitivity And Harmonisation Of Results Between Different Laboratories
  • Aliquots Of A Single Dna Preparation Were Analysed At Three Scandinavian Laboratories
  • The Developed Real-Time Pcr Assay Was Compared To Culturing By Analysing 126 Samples
  • And To A Conventional Pcr Method By Analysing 224 Samples
  • A Selection Of Pcr-Products Was Cloned And Sequenced In Order To Verify That They Had Been Identifie
  • Results: The Developed Assay Had A Detection Limit Of 3
  • 9 Fg Of D
  • Nodosus Genomic Dna
  • This Result Was Obtained At All Three Laboratories And Corresponds To Approximately Three Copies Of
  • Nodosus Genome Per Reaction
  • The Assay Showed 100% Inclusivity And 100% Exclusivity For The Strains Tested
  • The Real-Time Pcr Assay Found 54
  • 8% More Positive Samples Than By Culturing And 8% More Than Conventional Pcr
  • Conclusions: The Developed Real-Time Pcr Assay Has Good Specificity And Sensitivity For Detection Of
  • Nodosus
  • And The Results Are Easy To Interpret
  • The Method Is Less Time-Consuming Than Either Culturing Or Conventional Pcr

Abstract

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories - DTU Orbit (26/04/14) Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories - DTU Orbit (26/04/14) Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories Frosth, S., Slettemeås, J. S., Jørgensen, H. J., Angen, Ø. & Aspán, A. 2012 In : Acta Veterinaria Scandinavica. 54, 1, p. 6 Publication: Research - peer-review › Journal article – Annual report year: 2012

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