A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification

Prorocentrum donghaiense is the most common bloom-forming species in the East China Sea, causing serious damage to regional marine ecosystems, marine fisheries, public health, and aquatic environment. To minimize fisheries losses caused by this harmful alga, a simple and accurate detection method need to be developed to provide adequate early warning for P. donghaiense blooms. In this study, we report the development and application of reverse transcription-coupled loop-mediated isothermal amplification (RT-LAMP) in the field detection of P. donghaiense. The partial large subunit rDNA (LSU D1-D2), small subunit rDNA, and internal transcribed spacers of P. donghaiense were first sequenced to design species-specific amplification primers. Primer set screen showed that the LSU D1-D2 was the best target for LAMP in terms of sensitivity and stability. The amplification conditions including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time, and temperature, were optimized. The specificity of RT-LAMP for P. donghaiense was also confirmed through tests using a few common harmful algae. RT-LAMP targeting RNA exhibited a detection limit of 0.6 cells, which is more sensitive than LAMP and PCR targeting DNA and reverse transcription-polymerase chain reaction (RT-PCR) targeting RNA. Finally, an improved protocol for natural samples was applied to the field material. The optimized detection protocol could be completed within 1 h. In addition, positive RT-LAMP results could be confirmed through the production of white magnesium pyrophosphate precipitate or through mixing the fluorescent dye GeneFinder (TM) with the amplification products. In summary, the established RT-LAMP is specific, sensitive, and rapid method that is promising for the field detection of P. donghaiense. (c) 2013 Elsevier B.V. All rights reserved.