| 摘要: |
| 本研究将环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)与横向流动试纸条(lateral flow dipstick, LFD)的可视化检测方法结合, 建立了扁浒苔(Ulva compressa)的LAMP-LFD快速检测技术。该方法以扁浒苔的内转录间隔区(ITS1-5.8S-ITS2)序列为检测靶标, 设计了3对特异性引物(其中, 上游内引物由生物素标记)和1条异硫氰酸荧光素标记的探针。结果表明, LAMP最适反应温度为63℃, 扩增时间为60 min, 从核酸扩增到LFD结果判读需70 min。利用LAMP-LFD可特异性检出扁浒苔, 对浒苔、曲浒苔、缘管浒苔和孔石莼等石莼属绿藻以及塔玛亚历山大藻、无纹环沟藻、东海原甲藻、锥状斯克里普藻和赤潮异弯藻等常见微藻的检测结果为阴性。该方法最低可检测到0.1 pg的扁浒苔基因组DNA, 是以UcoITS-F3和UcoITS-B3为特异性引物的PCR方法的100倍。对实际样品的检测结果表明, LAMP-LFD方法检测扁浒苔与传统的形态学观察的结果一致。因此, 该方法可快速、特异地检测出扁浒苔, 而且操作简单, 仪器设备依赖性低, 有潜力成为扁浒苔现场检测的常规技术手段。 |
| 关键词: 扁浒苔 环介导等温扩增 横向流动试纸条 内转录间隔区 检测 |
| DOI：10.11693/hyhz20150200048 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863计划),2012AA092001号;浙江省重大科技专项重点社会发展项目,2013C03045-1号。 |
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| RAPID DETECTION OF ULVA COMPRESSA BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COMBINED WITH LATERAL FLOW DIPSTICK |
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CHEN Xian-Feng1,2, ZHOU Qian-Jin1, WANG Rui-Na1, DUAN Wei-Jun2, MIAO Liang1, CHEN Jiong1
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1.School of Marine Science, NingboUniversity, Ningbo, 315211;2.Academy of Inspection and Quarantine, Ningbo, 315012
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| Abstract: |
| A rapid loop-mediated isothermal amplification (LAMP) method combined with a lateral flow dipstick (LFD) was developed to detect Ulva compressa.Three pairs of primers that specifically recognized 8 conserved regions of the internal transcribed spacer regions (ITS1-5.8S-ITS2) were designed and UcoITS-FIP, the forward inner primer, was biotinylated.In addition, a DNA probe was designed to specifically recognize the given region of ITS1-5.8S-ITS2, and it was labeled by fluorescein isothiocyanate (FITC).The optimized conditions for LAMP assay were recommended at 63℃ within 60 min and the whole course including nucleic acid amplification and visual detection by LFD was less than 70 min.The LAMP-LFD assay could specifically detect U.compressa and distinguish it from U.prolifera, U.flexuosa, U.ohnoi, U.linza and U.pertusa.And no positive results could be observed from several common microalgal species, i.e., Alexandrium tamarense, Gyrodinium instriatum, Prorocentrum donghaiense, Scrippsiella trochoidea, and Heterosigma akashiwo.The LAMP-LFD assay achieved a limit of detection of 0.1 pg genomic DNA of U.compressa, which is 100 times lower than that of PCR assay using primers UcoITS-F3/UcoITS-B3.U.compressa could be successfully detected from filed samples by LAMP-LFD, which is coincident with the results obtained by the traditional microscopic examination.Therefore, this rapid and specific method is wellsuited for the detection of U.compressa in water samples. |
| Key words: Ulva compressa loop-mediated isothermal amplification lateral flow dipstick ITS1-5.8S-ITS2 detection |
