Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beerspoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beerspoilage species of Pectinatus and cocci, including new species. Beer is a microbiologically stable beverage due to the presence

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... antibacterial alcohol and hop compounds, as well as low pH and the depletion of dissolved oxygen and nutrients. 1) Thus, only a limited number of bacterial species, principally belonging to the genera Lactobacillus, Pediococcus, Pectinatus and Megasphaera, are known to spoil beer. As described in our previous report, we divided these beer-spoilers into three groups, Lactobacillus (Gram-positive rods), Pectinatus (Gramnegative rods), and Pediococcus and Megasphaera (Gram-positive and Gram-negative cocci) on the basis of morphological and Gram-staining features, and constructed three sets of multiplex PCR methods to detect beer-spoilage species in each group. 2) These multiplex PCR methods were designated L. multiplex (Lactobacillus), P. multiplex (Pectinatus), and C. multiplex (cocci) respectively. 2) However, four new beerspoilage species, Lactobacillus backi, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, have recently been proposed. 3,4) Hence, we investigated the applicability of our multiplex PCR methods to determine whether these new species can be detected by them. In the event, among these new species, only L. backi was detected by the L. multiplex method. L. backi is described as a species closely related to Lactobacillus coryniformis, and, was found to have DNA sequences corresponding to the L. multiplex primers specific to L. coryniformis. On the other hand,