title: Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.
abstract: A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.
authors.affiliation: Lethbridge Laboratory, National Centres for Animal Disease, Canadian Food Inspection Agency, Township Road 9-1, Lethbridge, Alberta T1J 3Z4, Canada. oliver.lung@inspection.gc.ca
authors.forename: Oliver
authors.initials: O
authors.lastname: Lung
authors.forename: Mathew
authors.initials: M
authors.lastname: Fisher
authors.forename: Anne
authors.initials: A
authors.lastname: Beeston
authors.forename: Kimberley Burton
authors.initials: KB
authors.lastname: Hughes
authors.forename: Alfonso
authors.initials: A
authors.lastname: Clavijo
authors.forename: Melissa
authors.initials: M
authors.lastname: Goolia
authors.forename: John
authors.initials: J
authors.lastname: Pasick
authors.forename: William
authors.initials: W
authors.lastname: Mauro
authors.forename: Dirk
authors.initials: D
authors.lastname: Deregt
mesh_terms: D000818:Animals; D002417:Cattle; D029822:Enterovirus B, Human / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005536:Foot-and-Mouth Disease / Q000175:diagnosis / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D046228:Microarray Analysis / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D013552:Swine; D013555:Swine Vesicular Disease / Q000175:diagnosis / Q000821:virology; D014720:Vesicular Exanthema of Swine / Q000175:diagnosis / Q000821:virology; D017928:Vesicular exanthema of swine virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*