P141: Effect of amplicon size on the differentiation of viable and nonviable Dekkera bruxellensis using EMA and real-time PCR

Dekkera bruxellensis is a problem in alcoholic fermentations. It creates off flavors, lowers ethanol yields and causes stuck fermentations.  Because of its impact on industry, a number of rapid detection methods, such as real-time PCR, have been developed.   One problem with DNA based methods is their inability to differentiate viable from nonviable cells.  To overcome this drawback, we developed an ethidium monoazide bromide (EMA) assay in combination with real-time PCR (QPCR).  Using primers specific for D. bruxellensis, we are able to differentiate viable from nonviable cells.  However, in the process of developing this assay, a question arose:  does the size of the amplicon affect its efficacy?  When using our original primer set (79 bp product), the addition of EMA increased the Ct value (23.09 to 29.42) in an assay containing 100% heat killed cells. When a different primer set (236 bp product) was used, the addition of EMA increased the Ct value (23.75 to 37.1), suggesting a larger product may yield better results.  The increased Ct indicates that, with the addition of EMA, it was possible to eliminate nonviable cells from detection. To further study this question, we developed two sets of primers (416 bp product and 116 bp product).  The primers, specific to Dekkera, were constructed using sequence from the horizontal adenyl deaminase gene, with one primer set internal to the other and was used to test our theory.  From this initial work, it appears that the size of the fragment increases the effectiveness of the assay.