title: Shining a light on LAMP assays--a comparison of LAMP visualization methods including the novel use of berberine.
abstract: The need for simple and effective assays for detecting nucleic acids by isothermal amplification reactions has led to a great variety of end point and real-time monitoring methods. Here we tested direct and indirect methods to visualize the amplification of potato spindle tuber viroid (PSTVd) by loop-mediated isothermal amplification (LAMP) and compared features important for one-pot in-field applications. We compared the performance of magnesium pyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I, EvaGreen, and berberine. All assays could be used to distinguish between positive and negative samples in visible or UV light. Precipitation of magnesium-pyrophosphate resulted in a turbid reaction solution. The use of HNB resulted in a color change from violet to blue, whereas calcein induced a change from orange to yellow-green. We also investigated berberine as a nucleic acid-specific dye that emits a fluorescence signal under UV light after a positive LAMP reaction. It has a comparable sensitivity to SYBR Green I and EvaGreen. Based on our results, an optimal detection method can be chosen easily for isothermal real-time or end point screening applications.
authors: Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Wildau, Germany.
authors: Jens
authors: J
authors: Fischbach
authors: Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Wildau, Germany.
authors: Nina Carolin
authors: NC
authors: Xander
authors: Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Wildau, Germany.
authors: Marcus
authors: M
authors: Frohme
authors: Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Wildau, Germany.
authors: Jörn Felix
authors: JF
authors: Glökler
mesh_terms: D052160:Benzothiazoles; D001599:Berberine / Q000032:analysis / Q000737:chemistry; D003959:Diamines; D011756:Diphosphates / Q000032:analysis / Q000737:chemistry; D005452:Fluoresceins / Q000032:analysis / Q000737:chemistry; D007202:Indicators and Reagents; D017616:Magnesium Compounds / Q000032:analysis / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000032:analysis / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009696:Nucleic Acids / Q000032:analysis*; D009930:Organic Chemicals / Q000032:analysis / Q000737:chemistry; D011804:Quinolines; D014772:Viroids / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D011756:Diphosphates; D005452:Fluoresceins; D007202:Indicators and Reagents; D017616:Magnesium Compounds; D009282:Naphthalenesulfonates; D009696:Nucleic Acids; D009930:Organic Chemicals; D011804:Quinolines; D001599:Berberine; C098022:SYBR Green I; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate; C034732:magnesium pyrophosphate; C007740:fluorexon
keywords: EvaGreen; LAMP; SYBR Green I; berberine; calcein; hydroxynaphthol blue; loop-mediated isothermal amplification; potato spindle tuber viroid