title: Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment.
abstract: Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30°C for 3h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.
authors.affiliation: Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science (NIFS), Tongyeong 650-943, Republic of Korea. Electronic address: taegyupark@korea.kr.
authors.forename: Tae Gyu
authors.initials: TG
authors.lastname: Park
authors.affiliation: Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science (NIFS), Tongyeong 650-943, Republic of Korea.
authors.forename: Jin Joo
authors.initials: JJ
authors.lastname: Kim
authors.affiliation: Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science (NIFS), Tongyeong 650-943, Republic of Korea.
authors.forename: Won Jin
authors.initials: WJ
authors.lastname: Kim
authors.affiliation: Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science (NIFS), Tongyeong 650-943, Republic of Korea.
authors.forename: Kyoung Mi
authors.initials: KM
authors.lastname: Won
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics; D004141:Dinoflagellida / Q000235:genetics*; D004784:Environmental Monitoring / Q000379:methods*; D057097:Harmful Algal Bloom; D012335:RNA, Ribosomal / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012623:Seawater / Q000469:parasitology; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D012335:RNA, Ribosomal
keywords: Cochlodinium polykrikoides; Cyst; Dinoflagellate; Real-time RT-PCR; Red tide