The development of an antigen capture reverse transcription polymerase chain reaction method for foot-and-mouth disease virus antigen detection

Several protocols were developed and evaluated for the capture of foot-and-mouth disease (FMD) virus antigen on a microtitre plate to enable RNA extraction and reverse transcription to be carried out in the plate wells prior to PCR amplification of the cDNA product. One protocol enabled the FMD viral antigen from suspensions of clinical samples to be captured in the well by coating with a cocktail of type-specific guinea pig anti-FMD virus immune sera. The viral RNA was extracted by heating the plate for five minutes at 90C and was subsequently detected by PCR amplification with universal primer sets. In an alternative protocol, specific primer sets were able to serotype FMD viruses of types O, A, C and Asia 1 after individual wells were coated with homologous type-specific guinea pig anti-FMD virus immune serum. Both protocols were specific for FMD virus and their sensitivity was the same as that of the RT-PCR currently used to supplement the routine methods of ELISA and virus isolation in cell culture for diagnosis at the OIE/FAO World Reference Laboratory for FMD, Pirbright. This antigen capture (immunocapture) RT-PCR methodology is seen as a prototype to the development of antigen capture RT-PCR or PCR-ELISA methods involving combinations of labelled reagents such as biotin with streptavidin or digoxigenin with antidigoxigenin to enable the entire protocol to be carried out in a microtitre plate well. These systems could increase the sensitivity of the RT-PCR for FMD diagnosis and would have the additional advantage of being objectively analysed by use of an ELISA plate reader linked to a computer.