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

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.

Detection of koi herpesvirus (KHV) in healthy cyprinid seed stock.

Koi herpesvirus (KHV) disease is a lethal disease in common carp, an important food fish in Asian countries, the seed of which is used in restocking programs for freshwater fishery management. We inspected apparently healthy seed stock of common carp Cyprinus carpio L. and Siberian crucian carp Carassius auratus for the presence of KHV using PCR-based diagnostic tests as a part of a stock enhancement program from 2009 to 2010 in Korea. Consequently, KHV was detected from 24 of 232 inspections with yearly detection percentages of 5.2% in 2009 and 15.5% in 2010 using PCR primer sets for TK or SphI-5 as recommended by the OIE Manual of Diagnostic Tests for Aquatic Animals. Results indicate that the SphI-5 primer set was slightly more sensitive than the TK primer set, as shown by a higher detection rate. To determine the genotype of the KHV strains detected in this study, ORF40-specific PCR amplification was conducted, and the PCR products from 6 samples showed 100% nucleotide sequence identity with a Japanese strain (GenBank accession number AP008984) but not with US (DG657948) and Israeli strains (DG177346). This report conclusively demonstrated the presence of KHV in externally healthy seed of common carp and Siberian crucian carp, indicating a possible risk that subclinically infected seed stock can be released with a potential threat to wild populations.