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基于LAMP-LFD技术的有害赤潮藻东海原甲藻(Prorocentrum donghaiense)快检方法
邹怡欣, 黄海龙, 乔龙亮, 党晨阳, 庞建虎, 吕佩文, 王佳棋, 朱鹏
宁波大学海洋学院 宁波 315211
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℃、30min,较常规PCR扩增缩短约2h。结果表明:LAMP-LFD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。
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
DOI:10.11693/hyhz20170800208
分类号:X55
基金项目: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。
RAPID DETECTION OF PROROCENTRUM DONGHAIENSE BASED ON LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COMBINED WITH LATERAL FLOW DIPSTICK
ZOU Yi-Xin, HUANG Hai-Long, QIAO Long-Liang, DANG Chen-Yang, PANG Jian-Hu, LÜ Pei-Wen, WANG Jia-Qi, ZHU Peng
Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo 315211, China
Abstract:
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℃ for 30min, saving nearly 2h 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 47pg/μLof 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.
Key words:  Prorocentrum donghaiense  loop-mediated isothermal amplification (LAMP)  lateral flow dipstick (LFD)  quick detection
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