title: Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture.
abstract: Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.
authors.affiliation: Laboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan.
authors.forename: Hsiao-Che
authors.initials: HC
authors.lastname: Kuo
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Wang
authors.forename: Peng-Peng
authors.initials: PP
authors.lastname: Chen
authors.forename: Young-Mao
authors.initials: YM
authors.lastname: Chen
authors.forename: Hui-Ching
authors.initials: HC
authors.lastname: Chuang
authors.forename: Tzong-Yueh
authors.initials: TY
authors.lastname: Chen
mesh_terms: D000825:Animal Structures / Q000821:virology; D000818:Animals; D017756:Aquaculture; D001492:Bass / Q000821:virology*; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology*; D030381:Nodaviridae / Q000302:isolation & purification*; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013624:Taiwan; D014773:Virology / Q000379:methods*