Detection of foot-and-mouth disease virus from culture and clinical samples by reverse transcription-PCR coupled to restriction enzyme and sequence analysisMargarita Sáiza, Diana B. de la Morenaa, b, Esther Blancoa, José I. Núñeza, b, Rufino Fernándeza and José M. Sánchez-Vizcaínoa
a Centro de Investigación en Sanidad Animal, INIA, 28130 Valdeolmos, Madrid, Spain
b Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain
(Received 28 March 2002; accepted 23 August 2002)
A reverse transcription-PCR (RT-PCR) method is presented for the highly sensitive and specific detection of foot-and-mouth disease virus (FMDV). A primer pair flanking a region of the viral polymerase gene (3D) corresponding to the C-terminus of the protein was designed and a single step RT-PCR reaction was developed. The assay allowed the detection of viral RNA from a variety of animal samples and from a wide range of FMDV isolates of different origins and serotypes. The presence of an Ahd I restriction site within the amplicon in 96% of the isolates analyzed allowed an additional confirmation step of the positive reactions by a simple digestion yielding characteristic fragment sizes. The set of primers described here was suitable for direct sequencing of the PCR product (290 bp), and the nucleotide sequences corresponding to the SAT 1 and SAT 3 strains were determined. The segment amplified, when used in phylogenetic studies, allowed the clustering of SAT isolates and the rest of FMDV strains as two separate lineages.
Key words: foot-and-mouth disease / direct RT-PCR / restriction enzyme analysis / sequence analysis
Correspondence and reprints: Margarita Sáiz tel. (34) 91 6202300; fax: (34) 91 6202247;
© INRA, EDP Sciences 2003