摘要
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.
摘要译文
在先前的研究中，CCESS在环介导等温扩增（LAMP）的反应，从天然植物细胞或休息的有毒藻亚历山大藻和链状亚历山大藻的囊肿。在这项研究中，我们研究了4种方法从A.藻和A.单一休息囊肿提取DNAcatenella获得更稳定，更好的放大成功，并促进明确的检测采用LAMP法。除了比较4种不同的DNA提取方法，即（1）在Tris-EDTA（TE）缓冲液中十六烷基溴化缓冲液中煮沸，（2）加热至65℃，（3）在0.5％的Chelex缓冲液中煮沸，和（4），在5％的Chelex缓冲液中煮沸，我们还研究了需要同质化DNA的提取在每个方法之前粉碎休息囊肿。孢囊的均质化被认为是对DNA提取在所有4种方法是必不可少的。检测时间是显著短于5％的Chelex缓冲器比在其它缓冲液和扩增成功率为100％（65/65），指示DNA提取的重要性，并在5％的Chelex缓冲器在亚历山大LAMP的有效性。
Satoshi Nagai[a]; Keigo Yamamoto[b]; Naotugu Hata[c]; Shigeru Itakura[a]. 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[J]. Marine Genomics, 2012,7: 51–56