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

作　　者：王建艳 甄毓 米铁柱 于志刚 王国善 

机构地区：[1]College of Marine Life Sciences, Ocean University of China [2]College of Environmental Science and Engineering, Ocean University of China [3]Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China [4]College of Chemistry and Chemical Engineering, Ocean University of China [5]Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China

出　　处：《Chinese Journal of Oceanology and Limnology》2015年第4期974-987,共14页中国海洋湖沼学报（英文版）

基　　金：Supported by the National Basic Research Program of China(973 Program)(No.2011CB403602);the National Natural Science Foundation of China(No.41076085);the National Special Research Fund for Non-Profit Marine Sector(No.201205031)

摘　　要：The complicated life cycle ofAurelia 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 （mr 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 ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL 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.The complicated life cycle of A urelia 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 16 SrDNA(mt 16 SrDNA) regions were designed. Using a calibration curve constructed with plasmids containing the A urelia sp.1 mt 16 SrDNA fragment and a standard curve for planulae, the absolute number of mt 16 SrDNA 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 μg/μL BSA in the PCR reaction mixture was used to remove realtime PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak A urelia 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.

关 键 词：Aurelia sp. 1 16S rDNA planulae real-time PCR jellyfish blooms 

分 类 号：Q503[生物学—生物化学]