title: Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV).
abstract: In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.
authors.affiliation: Division of Virology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden. cuevas.julieta@inifap.gob.mx
authors.forename: Sandra
authors.initials: S
authors.lastname: Cuevas-Romero
authors.forename: Anne-Lie
authors.initials: AL
authors.lastname: Blomström
authors.forename: Arcelia
authors.initials: A
authors.lastname: Alvarado
authors.forename: Pablo
authors.initials: P
authors.lastname: Hernández-Jauregui
authors.forename: Francisco
authors.initials: F
authors.lastname: Rivera-Benitez
authors.forename: Humberto
authors.initials: H
authors.lastname: Ramírez-Mendoza
authors.forename: Mikael
authors.initials: M
authors.lastname: Berg
mesh_terms: D000818:Animals; D002460:Cell Line; D021983:Cyclophilins / Q000032:analysis / Q000235:genetics; D016679:Genome, Viral; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D019213:Rubulavirus / Q000235:genetics / Q000302:isolation & purification*; D019351:Rubulavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: C455099:P protein, Porcine rubulavirus; D010750:Phosphoproteins; D012367:RNA, Viral; D014764:Viral Proteins; D021983:Cyclophilins