Development of a quantification method of HcRNAV infectious bloom-forming dinoflagellate Heterocapsa circularisquama with a monoclonal antibody and PCR

2014 Fiscal Year Final Research Report

• Project/Area Number: 24580290
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General fisheries
• Research Institution: Fisheries Research Agency
• Principal Investigator: NAKAYAMA Natsuko 独立行政法人水産総合研究センター, 瀬戸内海区水産研究所, 研究員 (20612675)
• Co-Investigator(Kenkyū-buntansha): NAGASAKI Keizo 独立行政法人水産総合研究センター, 本部, 研究開発コーディネーター (00222175) ; HAMAGUCHI Masami 独立行政法人水産総合研究センター, 瀬戸内海区水産研究所, 主幹研究員 (60371960)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: mRT-PCR / HcRNAV / ELISA / ヘテロカプサ

Outline of Final Research Achievements

We established the Enzyme-linked Immunoadosorbent Assay (ELISA) system and the multiplex real-time reverse transcription PCR (mRT-PCR) system for quantifying a single-stranded RNA virus (HcRNAV) that causes a lytic infection in bloom-forming dinoflagellate Heterocapsa circularisquama, and checked its availability to environmental samples. In mRT-PCR, two primer-probe sets targeting separate conserved regions in the HcRNAV genome allowed highly specific detection of HcRNAV. The accuracy of the system was tested using three typical HcRNAV strains by comparing the enumeration results of the mRT-PCR method and two conventional methods, transmission electron microscopy (TEM) and a most-probable-number (MPN) assay. As a result, estimates obtained via the mRT-PCR method were consistent with those of TEM, indicating that it is a useful method for quantifying HcRNAV. In the ELISA, a monoclonal and polyclonal antibody established became possible to specifically detect the HcRNAV in situ.

Free Research Field

藻類ウイルス学、赤潮科学