title: Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.
abstract: A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.
authors.affiliation: Virology Division, Kimron Veterinary Institute, P.O. Box 12, Beit-Dagan 50250, Israel. stramy@int.gov.il
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Stram
authors.forename: Larisa
authors.initials: L
authors.lastname: Kuznetzova
authors.forename: Merisol
authors.initials: M
authors.lastname: Guini
authors.forename: Arie
authors.initials: A
authors.lastname: Rogel
authors.forename: Ruth
authors.initials: R
authors.lastname: Meirom
authors.forename: Dalia
authors.initials: D
authors.lastname: Chai
authors.forename: Hagai
authors.initials: H
authors.lastname: Yadin
authors.forename: Jackob
authors.initials: J
authors.lastname: Brenner
mesh_terms: D000818:Animals; D001483:Base Sequence; D002043:Bunyaviridae / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D017931:DNA Primers; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase