Detection of culturable and viable but non-culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide andhorA-specific polymerase chain reaction

Current methods of detecting beer spoilage lactic acid bacteria (LAB) are time-consuming and do not differentiate between viable and non-viable bacteria. In this study, a combination of the conventional polymerase chain reaction (PCR) and propidium monoazide (PMA) pretreatment has been described to circumvent the disadvantages. The horA-specific PMA-PCR described here identifies beer spoilage LAB based not on their identity, but on the presence of a gene that is shown to be highly correlated with the ability of LAB to grow in beer. The results suggest that the use of 20 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable, but putatively non-culturable (VPNC) Lactobacillus acetotolerans. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. acetotolerans cells was 1.5 µg/mL. The detection limit of established PMA-PCR assays was found to be 100 VPNC cells/reaction for the horA gene. Furthermore, the horA-specific PMA-PCR assays were subjected to 18 reference strains, representing 100% specificity with no false positive amplification observed. In conclusion, the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for the detection of potential beer spoilage LAB and efficiently discriminates between live and dead cells. Copyright © 2015 The Institute of Brewing & Distilling