title: Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.
abstract: BACKGROUND
  The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.
  
  
  RESULTS
  In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.
  
  
  CONCLUSIONS
  The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.
authors.affiliation: Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
authors.forename: Sören
authors.initials: S
authors.lastname: Hansen
authors.affiliation: Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Schäfer
authors.affiliation: Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
authors.forename: Kim
authors.initials: K
authors.lastname: Fechner
authors.affiliation: Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
authors.forename: Claus-Peter
authors.initials: CP
authors.lastname: Czerny
authors.affiliation: Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abd El Wahed
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012418:Ruminants / Q000382:microbiology; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial