USE OF REAL-TIME RT-PCR

Foot-and Mouth Disease virus (FMDV) is diagnosed by virus isolation test combined with antigen ELISA and recently one step RT-PCR. Use of fluorogenic RT-PCR is getting more and more importance for rapid detection of FMDV from clinical samples that cannot be processed by regular virus isolation tests and antigen ELISA. Quantitative real-time RT-PCR (qRT-PCR) combines Reverse Transcription with PCR amplification and detection of positive amplification using fluorescent probes like TaqMan (R) probe. In the present study, a TaqMan(R) probe based qRT-PCR was standardized and used for detection of FMDV genome from clinical samples including nasal secretions, plasma and oesophago-pharyngeal fluids (probang samples). The initial standardization was made using different serial dilutions of a FMDV isolate OTNN 24/84 (106.8 TCID50) and 102 virus could be identified using the TaqMan(R) qRT-PCR. RNA standards were synthesized in vitro from a plasmid containing 110 base pair of 3D region of a FMDV type O isolate.