Identification of Beer-Spoilage Microorganisms Using the Loop-Mediated Isothermal Amplification Method. Youichi Tsuchiya (1), Masahiro Ogawa, Yasukazu Nakakita, Yasunobu Nara, Hirotaka Kaneda, and Junji Watari, Frontier Laboratories of Value Creation, Sapporo Breweries Ltd. Shizuoka, Japan; and Harumi Minekawa and Takahiro Soejima, Eiken Chemical Co., Ltd., Tokyo, Japan. (1) Corresponding author. E-mail: <Yoichi.Tsuchiya@sapporobeer.co.jp>; Phone: +81-54-629-7984; Fax: +81-54-629-3144. J. Am. Soc. Brew. Chem. 65(2):77-80, 2007.

A new method for identifying beer-spoilage microorganisms was developed using a unique nucleic acid amplification method. Loop-mediated isothermal amplification (LAMP), developed by Eiken Chemical Co., Ltd., reacts under isothermal conditions and produces large amounts of DNA. The LAMP method requires a set of four specifically designed primers and a DNA polymerase with strand-displacement activity. The amplification products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops. After the LAMP reaction, white precipitates identified as magnesium pyrophosphate are found in the reaction mixture, which confirms that DNA was amplified. We designed primers specific to four representative beer-spoilage bacteria, optimized the conditions for LAMP, and developed an identification kit containing all four primers in one reaction mixture. The kit was used to identify isolated colonies. The kit is simple, specific, cost-effective, and applicable for practical use. Keywords: Beer-spoilage microorganisms, Identification, LAMP, Loop-mediated isothermal amplification, PCR