基于LAMP-LFD技术的有害赤潮藻东海原甲藻(Prorocentrum donghaiense)快检方法  

RAPID DETECTION OF PROROCENTRUM DONGHAIENSE BASED ON LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COMBINED WITH LATERAL FLOW DIPSTICK

作　　者：邹怡欣 黄海龙[1] 乔龙亮 党晨阳 庞建虎 吕佩文 王佳棋 朱鹏[1] ZOU Yi-Xin;HUANG Hai-Long;QIAO Long-Liang;DANG Chen-Yang;PANG Jian-Hu;LU Pei-Wen;WANG Jia-Qi;ZHU Peng(Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo 315211, China)

机构地区：[1]宁波大学海洋学院,宁波315211

出　　处：《海洋与湖沼》2018年第3期586-593,共8页Oceanologia Et Limnologia Sinica

基　　金：宁波市科技攻关项目;2017C110003号;浙江省公益技术应用研究;2017C33133号;国家现代农业产业技术体系建设专项资金项目;CARS-47号

摘　　要：以东海原甲藻的ITS序列(Internal transcribed spacer,ITS)为检测靶标,将生物素标记的环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)扩增产物与经异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针特异性杂交,并结合横向流动试纸条(Lateral flow dipstick,LFD)肉眼直接观察检测结果,建立了有害赤潮藻东海原甲藻(Prorocentrum donghaiense)的快速检测技术。经优化后的最适条件为63°C、30min,较常规PCR扩增缩短约2h。结果表明:LAMPLFD可特异性检出东海原甲藻,对常见赤潮藻检测结果均为阴性;其对东海原甲藻基因组DNA的检测最低限为47pg/μL,是常规PCR技术(以F3/B3为引物)的10倍。LAMP-LFD技术能高效、特异地检出东海原甲藻,仪器设备依赖性低,结果可视化,有望成为赤潮原因种检测监控的常规方法。On the basis of the loop-mediated isothermal amplification（LAMP） combined with a lateral flow dipstick（LFD）, we established a rapid method to detect Prorocentrum donghaiense. A DNA probe labeled by fluorescein isothiocyanate（FITC） and forward inner primer biotinylated were designed specifically to identify the target gene. The hybridization of biotinylated LAMP product and the FITC-labeled probe in combination with LFD（lateral flow dipstick） were able to be detected visibly. The optimization conditions were 63°C for 30 min, saving nearly 2 h if by PCR. The results show that the new method is highly specific, and no characteristic amplification was observed when taking other common red tide algae species as a template. The detection limit was 47 pg/μL of P. donghaiense genomic DNA, which is 10 times lower than the conventional PCR using primers Pd-F3/Pd-B3. Therefore, LAMP-LFD can detect P. donghaiense efficiently and specifically, depend less on the equipment, and presents results in good visualization; and it may become a conventional method to detect and monitor red tide.

关 键 词：东海原甲藻 环介导恒温扩增技术 横向流动试纸条 生物快检 

分 类 号：X55[环境科学与工程—环境工程]