Application of multiplex PCR to the detection of beer-spoilage bacteria

Multiplex PCR methods were developed to detect 12 species of beer-spoilage bacteria that belong to the genera Lactobacillus, Pectinatus, Pediococcus, and Megasphaera. For microbiological quality control in breweries, we divided these species into three groups and evaluated a multiplex PCR method to simultaneously detect beer-spoilage species belonging to each group. Subsequently, the primer designs and blending ratios for each multiplex PCR analysis were optimized to satisfy the performance required for practical applications by breweries. As a result, each PCR method showed sufficient specificity to the target species and exhibited identical sensitivity with corresponding species-specific simplex PCR methods. In addition, none of the three multiplex methods showed a false-positive reaction to the nonspoilage bacterial strains frequently detected in breweries. Furthermore, artificial positive control DNAs were designed from X-phage genomic DNA sequences to assure the performance of the PCR tests. Consequently, only one PCR test tube was required to ensure that all primer pairs in a multiplex PCR method were fully functional, instead of using multiple test tubes for separate confirmation of the respective primer pairs. Taken together, these multiplex PCR analyses enabled us to reliably and comprehensively detect beer-spoilage bacteria more easily and inexpensively.