Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

作者：Wang Jianyan; Zhen Yu^*; Mi Tiezhu; Yu Zhigang; Wang Guoshan

来源：Chinese Journal of Oceanology and Limnology, 2015, 33(4): 974-987.

DOI：10.1007/s00343-015-4091-0

• Aurelia sp 1
• 16S rDNA
• planulae
• real-time PCR
• jellyfish blooms

The complicated life cycle of Aurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay (SYBR Green I) to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA (mt 16S rDNA) regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp.1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number of planulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 mu g/mu L BSA in the PCR reaction mixture was used to remove real-time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay (SYBR Green I) developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

• 出版日期2015-7
• 单位中国海洋大学