title: Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East.
abstract: Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). In countries where FMD is endemic, identification of the serotypes of the causative virus strains is important for vaccine selection and tracing the source of outbreaks. In this study, real-time reverse transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two copies. The results illustrate the potential of tailored rRT-PCR tools for the detection and categorization of viruses circulating in the Middle East belonging to distinct subgroups of serotypes O, A and Asia-1. These assays can also overcome the problem of serotyping samples which are found positive by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world.
authors.affiliation: Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Reid
authors.affiliation: Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.
authors.forename: Valerie
authors.initials: V
authors.lastname: Mioulet
authors.affiliation: Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Knowles
authors.affiliation: Khorasan Razavi Central Veterinary Laboratory, Iran Veterinary Organization, Iran.
authors.forename: Nazeem
authors.initials: N
authors.lastname: Shirazi
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.affiliation: Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom. Electronic address: donald.king@pirbright.ac.uk.
authors.forename: Donald P
authors.initials: DP
authors.lastname: King
mesh_terms: D000818:Animals; D003429:Cross Reactions; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D008877:Middle East; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D065288:Serogroup; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods
keywords: FMDV multiplex real-time serotyping RT-PCR assay; FMDV real-time serotyping RT-PCR assay; FMDV serotyping; Foot-and-mouth disease virus