关键词: 叠氮溴乙锭, 聚合酶链式反应, 啤酒腐败菌, 短乳杆菌, 酒花耐受基因

Abstract: In this paper, a rapid method using conventional PCR after ethidium bromide monoazide (EMA) pretreatment is described for the detection of Lactobacillus brevis as a beer spoilage bacterium. The PCR amplification was carried out using the hop resistance gene horC as target gene and the genomic DNA from L. brevis as template. The results suggested that addition of EMA to a final concentration lower than 20 μg/mL for pretreatment did not strongly inhibit the PCR amplification of DNA derived from viable L. brevis cells, but it, when added to a final concentration of 1.0 μg/mL, completely inhibited the PCR amplification of DNA derived from 105 CFU/mL dead L. brevis cells. The detection limit (LOD) of EMA-PCR assay was found to be 104 CFU/mL beer sample for the horC gene. Moreover, the horC-specific EMA-PCR assay was applied to detect 13 strains of lactic acid bacteria, representing 100% specificity with no false positive amplification observed for five beer spoilage lactic acid bacteria and enabling discrimination between the live and dead cells. Overall, the use of horCspecific EMA-PCR allows for a substantial reduction in the rate of false-positive results for potential beer spoilage L. brevis.

Key words: ethidium bromide monoazide (EBM), polymerase chain reaction (PCR), beer spoilage bacteria, Lactobacillus brevis, horC