title: Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage.
abstract: An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.
authors.affiliation: National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Mie 516-0193, Japan. yuasa@fra.affrc.go.jp
authors.forename: Kei
authors.initials: K
authors.lastname: Yuasa
authors.forename: Jun
authors.initials: J
authors.lastname: Kurita
authors.forename: Morihiko
authors.initials: M
authors.lastname: Kawana
authors.forename: Ikunari
authors.initials: I
authors.lastname: Kiryu
authors.forename: Norihisa
authors.initials: N
authors.lastname: Oseko
authors.forename: Motohiko
authors.initials: M
authors.lastname: Sano
mesh_terms: D000818:Animals; D002347:Carps / Q000821:virology*; D005393:Fish Diseases / Q000821:virology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D006564:Herpesviridae / Q000145:classification / Q000302:isolation & purification* / Q000502:physiology; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D006358:Hot Temperature; D012333:RNA, Messenger / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014779:Virus Replication / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral