Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.
In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP.
National Research Institute of Fisheries and Environment of Inland Sea, Maruishi 2-17-5, Hatsukaichi, Hiroshima 739-0452, Japan. snagai@affrc.go.jp
Satoshi
S
Nagai
Keigo
K
Yamamoto
Naotugu
N
Hata
Shigeru
S
Itakura
D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D004247:DNA / Q000302:isolation & purification*; D004141:Dinoflagellida / Q000235:genetics* / Q000302:isolation & purification; D004492:Edetic Acid; D019015:Geologic Sediments / Q000382:microbiology*; D006358:Hot Temperature; D007564:Japan; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011137:Polystyrenes; D011145:Polyvinyls; D013045:Species Specificity; D014325:Tromethamine
D002593:Cetrimonium Compounds; D011137:Polystyrenes; D011145:Polyvinyls; D014325:Tromethamine; C006960:chelex; D004247:DNA; D004492:Edetic Acid; D000077286:Cetrimonium