title: A one-step duplex rRT-PCR assay for the simultaneous detection of grass carp reovirus genotypes I and II.
abstract: Hemorrhagic disease of grass carp, caused by grass carp reovirus (GCRV), leads to severe economic losses in the grass carp farming industry in China. GCRV has been divided into three genotypes based on genome sequence. Genotypes I and II (GCRV-1 and GCRV-II, respectively) are the dominant genotypes and co-infections of GCRV-I and GCRV-II are common in grass carp aquaculture. A one-step duplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay was developed for simultaneous detection of GCRV-I and GCRV-II. The PCR assay is suitable for early diagnosis of grass carp hemorrhagic disease and for epidemiological surveillance. The detection limit of the assay is 10 copies for both GCRV-I and GCRV-II, which is as high as single-target rRT-PCR and higher than conventional RT-PCR. No cross reactivity with other GCRV subtypes or other viruses was observed. One hundred and twelve samples from grass carp suspected of hemorrhagic disease were collected from South and Central China. Eleven samples were positive for GCRV-I by RT-PCR alone, and fourteen samples were positive by single-target and duplex rRT-PCR. Forty two samples were positive for GCRV-II by RT-PCR alone and forty seven samples were positive by single-target and duplex rRT-PCR. Mixed infections were found in eight samples when analyzed by RT-PCR alone and in ten samples analyzed by single-target and duplex rRT-PCR. The duplex rRT-PCR system provides a sensitive and specific method to detect and differentiate between GCRV-I and GCRV-II in a single sample. This rRT-PCR assay could be a useful tool for the routine diagnosis of these two viruses and for epidemiology studies in grass carp aquaculture.
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Hongru
authors.initials: H
authors.lastname: Liang
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Cun
authors.initials: C
authors.lastname: Liu
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Xinjian
authors.initials: X
authors.lastname: Song
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China.
authors.forename: Chunbin
authors.initials: C
authors.lastname: Shi
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China. Electronic address: wushuqin001@21cn.com.
authors.forename: Shuqin
authors.initials: S
authors.lastname: Wu
authors.affiliation: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, 510380, China. Electronic address: sunny_929@163.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D017756:Aquaculture; D002347:Carps / Q000821:virology*; D002681:China; D060085:Coinfection / Q000662:veterinary; D005393:Fish Diseases / Q000821:virology*; D005838:Genotype; D012087:Reoviridae / Q000235:genetics / Q000302:isolation & purification*; D012088:Reoviridae Infections / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*
keywords: Discrimination; GCRV; Quantification; rRT-PCR