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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

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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.

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Authors and Affiliations

  1. National Engineering Research Center of JUNCAO Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

    Xu-Cong Lv, Rui-Bo Jia, Jing-Hao Chen, Wen-Bin Zhou, Yan Li & Bin Liu

  2. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

    Xu-Cong Lv, Rui-Bo Jia, Jing-Hao Chen, Wen-Bin Zhou, Yan Li, Bin Liu, Shao-Jun Chen & Yu-Ting Tian

  3. College of Biological Science and Technology, Institute of Food Science and Technology, Fuzhou University, Xue Yuan Road, University Town, Fuzhou, Fujian, 350002, People’s Republic of China

    Xu-Cong Lv, Bing-Xin Xu, Yi-Ting Liang, Ping-Fan Rao & Li Ni

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  1. Xu-Cong Lv
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  2. Rui-Bo Jia
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  12. Li Ni
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Corresponding authors

Correspondence to Xu-Cong Lv or Li Ni.

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Funding

This work was financially supported by grants from the Project Funded by China Postdoctoral Science Foundation (2015M570549), Projects of Science and Technology of Fujian Education Dept., China (JA14107), and Scientific Research Start-up Funding of Fujian Agriculture and Forestry University (grant no. KXML2012A).

Conflict of Interest

Xu-Cong Lv declares that he has no conflict of interest. Rui-Bo Jia declares that he has no conflict of interest. Jing-Hao Chen declares that he has no conflict of interest. Wen-Bin Zhou declares that he has no conflict of interest. Yan Li declares that she has no conflict of interest. Bing-Xin Xu declares that she has no conflict of interest. Yi-Ting Liang declares that she has no conflict of interest. Bin Liu declares that he has no conflict of interest. Shao-Jun Chen declares that he has no conflict of interest. Yu-Ting Tian declares that he has no conflict of interest. Ping-Fan Rao declares that he has no conflict of interest. Li Ni declares that she has no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Highlights

• Novel species-specific PCR primers for S. fibuligera, R. oryzae, and M. purpureus were developed.

• The specificity of the species-specific PCR primers was confirmed by RT-qPCR.

• The established assays were applied to the traditional brewing of Hong Qu glutinous rice wine.

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Lv, XC., Jia, RB., Chen, JH. et al. 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. Food Anal. Methods 10, 161–171 (2017). https://doi.org/10.1007/s12161-016-0565-8

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