Rapid Detection of Karenia brevis (Dinophyceae) by Loop-mediated Isothermal Amplification (LAMP)

Abstract

Karenia brevis(Gymnodinium breve) is an unarmored,non-peridinin-containing dinoflagellate.It produces lipophilic brevetoxin that can harm many marine animals,eg.fish,birds and shellfish.As for human,brevetoxin can cause respiratory distress by inhalation and food poisoning by consumption of contaminated shellfish.Previous methods for the detection of K.brevis depend on microscopy analysis,which is time-consuming and requires a considerable amount of expertise and skill.Molecular methods have also been applied to detect K.brevis,e.g.real-time PCR and DNA hybridization assay.However,there are some inevitable disadvantages for the two methods,such as expensive reagents and equipment,or fussy approaches.Loop-mediated isothermal amplification(LAMP) is a specific nucleic acid amplification method that is easy to perform.The LAMP method can amplify nucleic acids under isothermal conditions at approximately 65 ℃.As a result,it allows for the use of simple and inexpensive reaction equipment.The identification system of microalgae K.brevis was successfully established in this study using LAMP method.The specificity and sensitivity were validated based on this detection system.Nine different algae were investigated to confirm the specificity of the LAMP for detecting K.brevis.The results showed that only K.brevis and the samples containing K.brevis were amplified;no amplification occurred in all the other samples including K.mikimotoi.Moreover,LAMP was able to detect template at a 100 to 10-3 dilution,whereas regular PCR could amplify the sample at only a 100 to 10-2 dilution.Therefore,the detection limit of the LAMP system was approximately 5.0×10-2 ng(50 pg) DNA,which was 10 times more sensitive than conventional PCR.LAMP products could be identified by adding SYBR Green I dyes or by the production of a white precipitate.This visual inspection has predominance because there is no need for gel electrophoresis and staining with ethidium bromide.So this method illustrates a novel application prospect for forecasting red-tides caused by K.brevis because of its high sensitivity,rapid reaction and the need for only simple equipment.