Reducing bias in complex microbial community analysis in shrimp based on propidium monoazide combined with PCR-DGGE

Microbial community plays a prominent role in food quality and safety, which was regarded as the major cause for food spoilage and foodborne disease. Pre-PCR propidium monoazide (PMA) treatment is an effective approach to exclude DNA amplification signal from dead cells, and application of this method in the analysis of bacterial community might help us better understand the diversity and distribution of viable bacteria in food. In this study, the effects of PMA treatment on bacterial community characteristics in shrimp were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting technique. The results showed that the microbial diversity was dramatically influenced by PMA treatment. During storage at 4 °C, Shannon–Wiener index and PCoA analysis indicated there were significant differences between bacterial diversity in samples treated with and without PMA. At the level of species, compared with samples without PMA treatment, Vibrio cholera, Exiguobacterium acetylicum, Aeromonas enteropelogenes, Eubacterium multiforme, Vibrio mimicus in PMA-treated samples disappeared during storage. When samples stored at 25 °C, PMA treatment has little influence on banding patterns in DGGE profiles. In conclusion, this study provides an effective tool for precisely monitoring the diversity of viable bacteria in food samples, especially at low temperature.     

Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

The main disadvantage of DNA-based quantification methods is their inability to distinguish between viable and non-viable cells, as dead cells may also retain a significant amount of DNA. To overcome this limitation, propidium monoazide (PMA) treatment was applied to effectively prevent amplification of DNA from dead cells. The objectives of this study were to develop and evaluate an accurate detection method that combines PMA staining with real-time qPCR to quantify viable dominant microorganisms during the traditional brewing of Hong Qu glutinous rice wine. Firstly, three methodological factors (concentration of PMA, incubation time and photoactivation time) were optimized for representative strains of different microbial species. For the tested microbial strains (Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum), a treatment of 25 μM PMA for 20 min incubation followed by 10 min photoactivation was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction for the compromised cells. Compared with qPCR without PMA treatment, results of PMA-qPCR showed that the optimized PMA pretreatment can effectively exclude the dead microbial cells without any negative effects on the intact cells. The concentrations of viable cells calculated by PMA-qPCR were comparable (the same order of magnitude was obtained) with those obtained by culture-based method. Finally, the developed PMA-qPCR methods were successfully applied to monitor the total bacteria, LAB, fungi, yeasts, M. purpureus, S. cerevisiae and L. plantarum during the traditional brewing of Hong Qu glutinous rice wine. Both of the total bacterial and fungal populations increased significantly at the early brewing stage, but they decreased at the later brewing stage. PMA-qPCR results revealed that the predominant microorganisms varied in different brewing phases. L. plantarum and S. cerevisiae grew significantly and became the predominant bacterial and fungal species at the final stage of traditional brewing process, respectively. This study standardized the PMA-qPCR parameters for dominant microorganisms associated with the traditional brewing of Hong Qu glutinous rice wine, which would show great value in scientifically understanding of the brewing mechanism and provide useful information for the selection of beneficial microbial strains to improve wine quality.