Shini Yang, Yanlin Liu,* Peixin Cao, Dongqing Ye, and Yi Qin
*College of Enology, Northwest A&F University, College of Enology, Northwest A&F University, 23 Xinong Road, Yangling, Shaanxi Province 712100, China, 712100, China (805272810@qq.com)

Brettanomyces bruxellensis is one of the major spoilage microorganisms in wines, responsible for off-odor. In recent years, B. bruxellensis contamination has become more common in China. Conventional wine treatments will not prevent contamination, partially due to its remarkable SO2 and ethanol tolerance compared with Saccharomyces cerevisiae. Thus, it is critical to detect it early during winemaking. Traditional methods such as differential medium or specific PCR amplification can detect B. bruxellensis, but such methods need time, trained technicians, and specialized equipment that may not be available to a winery. Therefore, a simple and quick way to detect the presence of B.bruxellensis in wine is desirable. We have developed a method to detect B. bruxellensis rapidly, based on loop-mediated isothermal amplification. The primers were designed to target on the sequence of 5.8S-ITS-18S and specifically amplify the DNA genome of B. bruxellensis. The method targets six specific regions, designed inside and outside of four primers by design software. The LAMP reaction system was optimized for dNTP concentration, Mg2+ concentration and reaction temperature. The results showed that 0.8 mmol/L dNTP, 3.0 mmol/L Mg2+, and reaction temperature 62oC is suitable. In addition, we adopted visual observation and analysis to verify whether a reaction occurs by white precipitate or fluorescence staining. The method was specific and could verify the presence of several known yeasts. The detection threshold of DNA was down to 15 pg/μL. Compared with traditional PCR, LAMP is simple to operate and low-cost. It does not rely on any special instrument or equipment to detect B. bruxellensis rapidly under practical production conditions.

Funding Support: Northwest A&F University, China