Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

Abstract

The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 μg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 μg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Selected Instrumental Techniques Applied in Food and Feed: Quality, Safety and Adulteration Analysis

“…Ma and coworkers [ 151 ] applied propidium monoazide prior to PCR analysis, allowing live/dead discrimination of bacteria, and used the method to detect beer spoilage bacteria. The intercalating dye covalently binds to the genomic DNA of damaged cell membranes, and once in this state, it cannot be amplified.…”

Horseradish Peroxidase Labelled-Sandwich Electrochemical Sensor Based on Ionic Liquid-Gold Nanoparticles for Lactobacillus brevis

“…Lactobacillus brevis is the most common bacteria isolated from spoilage beer, which causes more than half of beer spoilage problems. It is also one of the beer spoilage bacteria which has been studied deeply [ 4 , 5 , 6 ].…”

Reduction, Prevention, and Control of Salmonella enterica Viable but Non-culturable Cells in Flour Food

“…Therefore, the detection of S. enterica VBNC cells by resuscitation is infeasible. In recent years, combining propidium monoazide (PMA) treatment with nucleic acid amplification technologies has shown to be capable of rapidly detecting VBNC bacteria ( Wang et al, 2011 ; Xu et al, 2011a , b , 2012a , b , 2016a , b ; You et al, 2012 ; Liu et al, 2015 , 2017 ; Jiang et al, 2016 ; Lin et al, 2016 ; Miao et al, 2016 , 2017a , b , 2018 ; Ma et al, 2017 ; Wang et al, 2019 ).…”