title: The development and evaluation of cross-priming amplification for the detection of avian reovirus.
abstract: OBJECTIVE
  The aim of this study was to develop and evaluate cross-priming amplification (CPA) for the detection of avian reovirus (ARV).
  
  
  RESULTS
  Five specific primers were designed, on the basis of the σNS sequence of the S1133 ARV strain. Incubation temperature and primer concentrations were determined. The optimal incubation conditions in a water bath were 61.3°C for 45 min. No reverse transcription stage was required. The results were recorded under UV light illumination as a bright, greenish fluorescence in positive samples, and through the lack of this in negative controls and samples. Additionally, the gel electrophoresis performed during analysis showed the presence of ladder-like patterns, formed by hairpin-like CPA products. The developed CPA method was compared to reverse-transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Sensitivity of CPA was estimated using seven dilutions of standard S1133 strain and reached 0.05 log10 TCID50 ml(-1). RT-PCR sensitivity reached 2.5 log10 TCID50 ml(-1) and was 1000 times lower than for CPA, whereas real-time RT-PCR sensitivity reached 1.5 log10 TCID50 ml(-1). Analysis of 32 RNAs extracted from field specimens showed the presence of an ARVσNS fragment in 4 (12.5%) samples. Interestingly, the positive samples originated from flocks affected by Marek's disease (MD) or fowl adenovirus (FadV). RT-PCR was unable to detect ARV, due to its lower sensitivity. However, the real-time RT-PCR that was conducted confirmed the CPA study.
  
  
  CONCLUSIONS
  CPA is a very sensitive and rapid method, which allows ARV detection using simple laboratory equipment.
  
  
  CONCLUSIONS
  This is the first report on the application of the CPA method for detection of ARV, using simple laboratory equipment.
authors.affiliation: Department of Poultry Viral Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: G
authors.initials: G
authors.lastname: Woźniakowski
authors.forename: J S
authors.initials: JS
authors.lastname: Niczyporuk
authors.forename: E
authors.initials: E
authors.lastname: Samorek-Salamonowicz
authors.forename: A
authors.initials: A
authors.lastname: Gaweł
mesh_terms: D000818:Animals; D002645:Chickens / Q000821:virology; D017931:DNA Primers; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D030101:Orthoreovirus, Avian / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D017931:DNA Primers
keywords: CPA, detection; avian reovirus; cross-priming amplification; isothermal amplification