Development of Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Assays for Monitoring Saccharomycopsis fibuligera, Rhizopus oryzae, and Monascus purpureus During the Traditional Brewing of Hong Qu Glutinous Rice Wine

Abstract

In this study, reverse transcription quantitative real-time PCR (RT-qPCR) assays using species-specific primers were developed for the quantification of the three major fungal species (Saccharomycopsis fibuligera, Rhizopus oryzae, and Monascus purpureus) during the traditional brewing of Hong Qu glutinous rice wine. Species-specific primers (Sfi-F/Sfi-R, Ro-F/Ro-R, and Mp-F/Mp-R) targeting the ITS-5.8S rRNA gene and the beta-tubulin gene sequences were designed and their specificity was evaluated by RT-qPCR with the total RNA from fungal species closely related to the traditional brewing of Hong Qu glutinous rice wine. Results of RT-qPCR using species-specific primers showed 100 % inclusivity (positive signal in the presence of target complementary DNA (cDNA)) and 100 % exclusivity (negative signals in the presence of non-target template). The specificity of each primer set was also tested when the target cDNA was mixed with non-target cDNA, and results showed that there was no significant difference (P > 0.05) in threshold cycle (Ct) values obtained from pure target cDNA and target cDNA mixed with non-target cDNA. The detection limits of the established qPCR assays were determined to be 101 gene copies/μL for S. fibuligera and 102 gene copies/μL for R. oryzae and M. purpureus, respectively. Good correlations were obtained for the tested species between 102 and 108 gene copies/μL by qPCR analysis. Finally, the established RT-qPCR assays were applied to quantify viable S. fibuligera, R. oryzae, and M. purpureus during the traditional brewing of Hong Qu glutinous rice wine. This study standardized the RT-qPCR assays for dominant fungi associated with the traditional brewing of Hong Qu glutinous rice wine, and it also proved the usefulness of RT-qPCR assays for the quantification of live cells in multi-species samples. Results would show great value in scientifically understanding of the brewing mechanism of Hong Qu glutinous rice wine.