Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues

Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has importa nt consequences for FMD control during both prophylactic vaccination progra mmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from t he disease. The rapid and effective control of FMD in pigs is especially im portant in regions of high pig density since infected pigs have the potenti al to generate plumes of airborne virus and spread infection beyond the imm ediate control area. Increased knowledge of the kinetics of FMDV replicatio n in pigs, especially in their respiratory tracts, could create opportuniti es for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT -PCR assay specific for the IRES (internal ribosome entry site) sequence of the O-1 Kaufbeuren/Lausanne strain of FMDV has been developed for this pur pose. The assay had a sensitivity of 0.1 TCID50/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significan t shortcomings when applied to a set of 16 anatomically highly diverse soli d tissues: (i) tissue differences in housekeeping gene expression caused er rors of up to 60-fold in estimated FMDV concentrations; and (ii) variabilit y in total RNA yields caused unpredictable saturation of RT reactions, whic h in turn caused errors of up to 250-fold in the estimated FMDV concentrati on. A novel quantitation strategy, designated the C-t(min) method, was deve loped to overcome these problems. The C-t(min) method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitat ed total RNA, spanning the optimum for the RT-PCR system used. The new meth od was influenced minimally by any tissue-specific RT-PCR inhibitors and wa s used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important ro le in the replication of FMDV in pigs than was thought previously. (C) 2001 Elsevier Science B.V. All rights reserved.