Rapid Detection of Lactic Acid Bacteria in Fermenter Samples Using a Nested Polymerase Chain Reaction

Abstract

Polymerase chain reaction (PCR) is a technique that can be used to detect extremely small amounts of target DNA. We have examined the reactivity of two primers that bind exclusively with lactic acid bacteria including species of Pediococcus, Lactobacillus, and certain Leuconostoc species. Neither yeast nor non-lactic acid bacteria react with these primers. However, the presence of greater than 3 × 104 yeast cells inhibited bacterial detection when performing a standard specific PCR reaction. This yeast interference was overcome when a nested PCR protocol was adopted. Nested PCR consists of two rounds of PCR with two distinct primer sets. The first round amplifies a fragment of DNA that contains within its sequence the primer sites for the second round of PCR. With the nested PCR, low levels of bacteria were detected in the presence of 108 yeast cells, which is well within the range required to screen fermenter samples for contamination. The entire reaction could be completed within an eight-hour work shift. This procedure assesses bacterial contamination in a few hours (rather than several days) before the end of fermentation and the decision point to collect yeast for re-pitching.