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

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.