DEVELOPMENT AND VALIDATION OF A ONE-STEP SYBR GREEN I - BASED REAL-TIME RT-PCR ASSAY FOR THE DETECTION AND QUANTIFICATION OF AVIAN METAPNEUMOVIRUS SUBTYPE A AND B

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like Avian Metapneumovirus (AMPV), where effective control benefits from a continuously updated knowledge of the epidemiological situation. Other real-time RT-PCRs have been published based on highly specific fluorescent-dye labeled probes but they have high initial cost, a complex validation and a marked susceptibility to the genetic variability of their target sequence. With this in mind we developed and validated a SYBR Green I based qRT-PCR for the detection of the two more prevalent AMPV subtypes (i.e subtype A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 TCID50/mL for both A and B subtypes. The high efficiency and linearity between viral titre and Cp displayed for both subtypes make it suited for viral quantification. Reaction conditions optimization and implementation of melting curve analysis guaranteed the high specificity of the assay. The stable Tm difference between the two subtypes, revealed the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific and rapid tool enabling contemporaneous detection, quantification and discrimination of AMPV subtype A and B.