DEVELOPMENT OF A NOVEL LAMP-LFD METHOD FOR RAPID DETECTION OF ULVA PERTUSA
Author(s): ZHOU Qian-Jin, CHEN Xian-Feng, CAI Yi, DUAN Li-Jun, DUAN Wei-Jun, MIAO Liang, CHEN Jiong, School of Marine Science, Ningbo University, Academy of Inspection and Quarantine
Pages: 380-389
Year: 2016 Issue: 2
Journal: Oceanologia et Limnologia Sinica
Keyword: Ulva pertusa; internal transcribed spacer; loop-mediated isothermal amplification; lateral flow dipstick; detection;
Abstract: We developed a novel LAMP-LFD method for rapid detection of Ulva pertusa based on loop-mediated isothermal amplification（LAMP） integrated with visual detection by a lateral flow dipstick assay（LFD）. Six primers were designed according to the conserved regions of internal transcribed spacer（ITS）（among the primers, the forward inner primer was biotinylated） and used in the LAMP assay. In addition, a specific probe was designed based on the conserved sequence amplified by both outer primers, and labeled by fluorescein isothiocyanate（FITC）. The biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected via LFD. The optimized LAMP assay to detect U. pertusa was performed at 63 °C for 50 min, and for only 60 min when visualization via LFD is incorporated. The results demonstrate that LAMP-LFD could specifically detect U. pertusa and no characteristic amplification was observed when using genomic DNA of other 9 common algal isolates responsible for green tide or red tide. The detection limit using genomic DNA of U. pertusa was 3.04×10–2 pg/μL, which was 1000 times lower than that of the conventional PCR method using both outer primers. U. pertusa could be successfully detected from filed samples by LAMP-LFD, which is coincident with the results obtained by the traditional microscopic examination. Therefore, the LAMP-LFD method is specific, sensitive, and easy, showing great potential in the rapid detection and routine monitoring of U. pertusa in the eastern coast of China.
Pages: 380-389
Year: 2016 Issue: 2
Journal: Oceanologia et Limnologia Sinica
Keyword: Ulva pertusa; internal transcribed spacer; loop-mediated isothermal amplification; lateral flow dipstick; detection;
Abstract: We developed a novel LAMP-LFD method for rapid detection of Ulva pertusa based on loop-mediated isothermal amplification（LAMP） integrated with visual detection by a lateral flow dipstick assay（LFD）. Six primers were designed according to the conserved regions of internal transcribed spacer（ITS）（among the primers, the forward inner primer was biotinylated） and used in the LAMP assay. In addition, a specific probe was designed based on the conserved sequence amplified by both outer primers, and labeled by fluorescein isothiocyanate（FITC）. The biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected via LFD. The optimized LAMP assay to detect U. pertusa was performed at 63 °C for 50 min, and for only 60 min when visualization via LFD is incorporated. The results demonstrate that LAMP-LFD could specifically detect U. pertusa and no characteristic amplification was observed when using genomic DNA of other 9 common algal isolates responsible for green tide or red tide. The detection limit using genomic DNA of U. pertusa was 3.04×10–2 pg/μL, which was 1000 times lower than that of the conventional PCR method using both outer primers. U. pertusa could be successfully detected from filed samples by LAMP-LFD, which is coincident with the results obtained by the traditional microscopic examination. Therefore, the LAMP-LFD method is specific, sensitive, and easy, showing great potential in the rapid detection and routine monitoring of U. pertusa in the eastern coast of China.
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