배양을 통하지 않고 혼합 현장시료에서 섬모충류 특히 염기서열을 감지하는 방법

초록

Ciliates (phylum Ciliophora) are one of the better-characterized group in the protozoans. Ciliates are important ecological bridge as first level consumers and second level producers in marine aquatic environment. They are considered to be valuable indicators for assessing environmental changes because they can be found in any kind of marine habitats with high species diversity and population density. Furthermore, they can react very sensitively to environmental factors such as pollutants owing to their small body size as one cell/one individual animals. The ciliates, however, have been applied restrictively as bio-indicator for marine environment analysis because sometimes identification of species and measurement of abundance of these tiny animals are considered as difficult and laborious tasks by most ecologists. Recently the field application of species-specific molecular markers for identification of marine animals have been increased. Most of attempts for molecular marker development, however, have been restricted to large sized animals such as crustaceans, annellids, and mollusks because laborious pure cultures of species were essential to obtain sequences from small sized animals like protozoans. Recently we obtained ciliate specific sequences from mixed field samples without pure cultures. Two ciliate specific primers were designed from 18S rDNA sequence alignment including diverse eukaryotes of fungi, algae, diatoms, and many protozoans. PCR products were cloned into pGEM-T easy vector and several white colonies were selected randomly and digested by TaqI restriction enzyme. Digested PCR products were grouped into 14 according to length variations and selected clones of each group were sequenced. Ten sequences out of fourteen were confirmed as ciliate specific (71%). Take into consideration that only 1-5% species are ciliates in mixed marine samples, the specificity of our primers for ciliates are very high