Development of a one-step Real Time RT-PCR assay for the detection, quantitation and differentiation of Avian Metapneumovirus subtype A and B

Direct diagnosis of avian Metapneumovirus (AMPV) infections rely on molecular techniques more than on virus isolation due to the fastidious nature of the virus. Six real-time reverse transcription PCR (RRT-PCR) protocols for the detection and differentiation of AMPV subtype A and B were developed in N (two tests), F, SH (two tests) and G genes. Five assays used SYBR Green I as detection system, and one molecular beacon probes. Specificity was evaluated using various AMPV strains, Newcastle disease, Infectious laryngotracheitis and Infectious bronchitis viruses. All tests were able to detect AMPVs and failed to detect non-AMPV viruses, and five out of six of them were also able to discriminate between AMPV A and B subtypes. Sensitivity was determined using serial dilutions of RNA from AMPV of both subtypes. The best results in terms of specificity and sensitivity was given by the RRT-PCR protocol designed in the SH gene. This new assay was later compared with a well-established molecular assay, an RT Nested-PCR based on the G gene