Fluorescence-based multiplex real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth...

This study reports the development of distinct fluorescence-based multiplex rRT-PCR arrays, which are comprised of unique primer pairs that are statistically modeled from publicly available sequences, for overall foot-and-mouth disease virus and serotype-specific detection. Performance of the multiplex pan FMDV (MpFMDV) array was compared to the 5' UTR and two 3D(pol) assays, following tests on the Miniopticon (BioRad) and 7900HT SDS (ABI) platforms. The MpFMDV array and a recently developed alternative 3D(pol) assay correctly identified all 31 isolates that represented all seven serotypes, including 10 isolates reported previously to be undetectable by both OIE recommended assays (5' UTR and 3D(pol) assay). Statistically significant differences in mean C(T) values were observed with both platforms. With the Miniopticon, the MpFMDV assay detected FMDV at 9.86 and 2.67 cycles less than the 5' UTR and one of the 3D(pol) assay, respectively; whereas with the 7900HT SDS, the MpFMDV assay detected FMDV at 7.11, 4.71, and 2.33 cycles less than the 5' UTR and both 3D(pol) assays, respectively. The MpFMDV assay was more sensitive than the 5' UTR assay, which had a higher mean endpoint dilution of 1.3 log(10). With the exception of the serotype A and C multiplex arrays, the multiplex serotype-specific arrays correctly identified all isolates from five of seven serotypes (O, Asia 1, SAT 1, 2, and 3). Results presented in this study provide proof-of-principle and bench validation for the primer design model, and for the ability of multiplex arrays to accurately detect FMDV and to provide some degree of serotype discrimination of FMDV.