The development and evaluation of cross-priming amplification for the detection of avian reovirus

AIMS: The aim of this study was to develop and evaluate cross-priming amplification (CPA) for the detection of avian reovirus (ARV). METHODS AND 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. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the application of the CPA method for detection of ARV, using simple laboratory equipment.