Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

• Published in: Harmful algae 2016-12, Vol.60, p.36-44
• Main Authors: Park, Tae Gyu, Kim, Jin Joo, Kim, Won Jin, Won, Kyoung Mi
• Format: Article
• Language: English
• Subjects: Cochlodinium polykrikoides; Cyst; Dinoflagellate; Dinophyceae; Real-time RT-PCR; Red tide
• ISSN: 1568-9883; 1878-1470
• DOI: 10.1016/j.hal.2016.10.005

•Use of real-time RT-PCR for Cochlodinium polykrikoides cyst survey in Korean sediments.•Detection accuracy was improved by removing RNA from dead vegetative cells in sediments.•Cysts were detected only at a few sites where high-density blooms occur every year. 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.