Development of Real Time RT-PCR tests for detection and differentiation of Avian Metapneumovirus

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. In five assays SYBR Green I was used as detection system, and in one, molecular beacon probes. Specificity was evaluated using various AMPV strains and other avian respiratory viruses such as Newcastle disease virus, Infectious laryngotracheitis virus 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 extracted from AMPV of both subtypes. The best results in terms of specificity and sensitivity were given by the RRT-PCR protocol developed using a set of primers designed in the SH gene.