Diagnosis of Foot-and-Mouth disease virus by automated RT-PCR

Automated 5' nuclease probe-based (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) procedures using a MagNA Pure LC were evaluated for foot-and-mouth (FMD) virus diagnosis during the United Kingdom (UK) 2001 epidemic. Epithelial suspensions (ES), serum or whole blood, milk and oesophageal-pharyngeal fluid (“probang”) samples submitted to the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright, were tested as well as supernatant fluids following inoculation of cell cultures with ES. New programmes have been inserted into the software of the MagNA Pure LC to improve the extraction of nucleic acids from test samples and controls and to increase the speed, flexibility, capacity and reproducibility of the RT and PCR procedures without harming the assay sensitivity. Like the initial programmes for automation, the new programmes enabled definitive diagnostic results to be achieved on first passage cell culture supernatant fluids but a 96-well PCR assay can now be performed by 2 people within an extended working day. Our results indicate that our real-time automated RT-PCR could be recommended instead of virus isolation during an outbreak to accelerate FMD diagnosis. The positive-negative acceptance criteria for the testing of probangs by automated RT-PCR is under consideration. Preliminary results from experimentally infected animals show that the virus can be detected in probangs but different assay acceptance criteria to that based on the testing of ES and cell culture supernatant fluids will have to be used.