Appendix 25
Diagnosis of Foot-and-Mouth disease virus by automated RT-PCR Scott M. Reid, Nigel P. Ferris, Geoffrey H. Hutchings and Soren Alexandersen Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom
Abstract 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 realtime 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. 1. Introduction If laboratory investigations are to play a part in FMD diagnosis following the control policies introduced by the UK government during the 2001 epidemic then the time to perform tests will have to be considerably accelerated. In the WRL for FMD, Pirbright, an evaluation is being made of the application of automated RT-PCR technology for the rapid and accurate diagnosis of the disease. Automated RT-PCR procedures for the diagnosis of FMD virus using tissue epithelium, serum or whole blood, milk and probang samples were initially evaluated on samples submitted to the WRL for FMD during the UK 2001 epidemic and from animals experimentally infected with the FMD virus serotype O UK 2001. A primer/probe set designed for the intended detection of all 7 serotypes of FMD virus was used (Reid et al., 2002) and the results of this evaluation were directly compared to the routine diagnostic tests of ELISA and virus isolation in cell culture (Reid et al., 2001a; 2001b). Automated programmes increased the speed and capacity of the RTPCR assays by enabling larger panels of clinical samples (together with positive and negative controls) to be tested simultaneously by RT-PCR. In an extended working day, one operator 203
