Development of a real-time reverse transcription PCR assay for detection of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula).

Abstract

AIMS: To develop a quantitative reverse transcription PCR (RT-qPCR) assay for detection of the putative wobbly possum disease (WPD) virus and to apply this test to investigate the viral load in archival tissues from past WPD transmission studies. METHODS: The real-time assay was developed as a two-step RT-qPCR in a SYBR green format and validated using serial dilutions of a linearised plasmid containing target DNA. The copy number values were normalised to the amount of RNA in each reverse transcription reaction and presented as viral copies/pg RNA. The viral load was determined in archival samples from animals that had received inoculations of infectious WPD tissue suspensions. Thirty sam..