31. Evaluation of automated RT-PCR systems to accelerate FMD diagnosis

Abstract

Automated 5'-nuclease probe-based RT-PCR procedures have been evaluated for FMD diagnosis using tissue epithelium, serum/blood, milk and oesophageal-pharyngeal fluid (“probang”) samples submitted to the OIE/FAO World Reference Laboratory for Foot-andMouth Disease (WRL for FMD), Pirbright, from the current United Kingdom (UK) epidemic and from experimentally infected animals. The RT-PCR results were directly compared to the results of the routine diagnostic tests of ELISA and virus isolation in primary calf thyroid cell culture. A MagNA Pure LC was programmed to automate the nucleic acid extraction and reverse transcription (RT) procedures with PCR amplification carried out by 5'-nuclease probe-based assay on a 5700 thermal cycler. The PCR amplification was also automated later in the evaluation. Automated RT-PCR successfully detected FMD virus in suspensions of tissue epithelium (ES) and in cell cultures following their inoculation with ES. The results of RT-PCR and virus isolation/ELISA on original serum/blood and milk samples were in broad agreement but the positive-negative acceptance criteria for the RT-PCR testing of probangs has to be fully optimised. This evaluation, however, demonstrated that automated RT-PCR has the potential to accelerate FMD diagnosis as positive results were achieved on first passage cell culture supernatant fluids from samples not showing an observable cytopathic effect until second passage.