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

Abstract

The aim of this study was to develop and evaluate cross-priming amplification (CPA) for the detection of avian reovirus (ARV). 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. CPA is a very sensitive and rapid method, which allows ARV detection using simple laboratory equipment. This is the first report on the application of the CPA method for detection of ARV, using simple laboratory equipment.