建立实时荧光定量PCR(real-time fluorescence quantitative PCR, RT-qPCR)方法对大曲中3种高温放线菌(Thermoactinomyces vulgaris、T.intermedius和T.daqus)进行特异性定量检测。根据T.vulgaris、T.intermedius、T.daqus全基因组中特异性单拷贝核心基因recA、mdH、gyrA为参考序列分别设计了一对可扩增278、233、291 bp片段的引物;经特异性、有效性、准确性实验显示,在常见的13种白酒酿造微生物中,引物TV、TI、TD分别可特异性检测T.vulgaris、T.intermedius、T.daqus,检测范围为2.82~8.82 lg copies/μL,加标回收率95%~105%。进一步对大曲发酵过程样品检测,结果显示,T.vulgaris含量在呈现波动趋势,在第5天达到最高,为(6.06±0.02) lg copies/g;T.intermedius含量呈现先上升后下降的趋势,在第7天达到最高,为(6.33±0.13) lg copies/g;T.daqus 含量在0~12 d呈现波动趋势,在12~14 d下降,随后一直上升,发酵结束时含量最高,为(7.62±0.02) lg copies/g。该研究建立的RT-qPCR方法可对大曲发酵过程中3种高温放线菌进行特异性鉴定和快速定量,为进一步监测高温放线菌在白酒酿造过程中的功能提供了方法。
To quickly and accurately detect three species of Thermoactinomyces (Thermoactinomyces vulgaris, T.intermedius, and T.daqus) in the Daqu fermentation process, a real-time fluorescence quantitative PCR method was established by designing specific primers.In this study, a pair of primers were designed using recA of T.vulgaris as a reference sequence for a 278 bp amplification product.Using mdH, a single copy core gene of T.intermedius, as a reference sequence, a pair of primers that can amplify 233 bp were designed.A pair of primers with 291 bp amplification were designed based on gyrA, a single copy core gene of T.daqus.The specificity, effectiveness, and accuracy tests showed that primers TV, TI, and TD could specifically detect T.vulgaris, T.intermedius, and T.daqus, respectively, and the detection range was 2.82 lg copies/μL-8.82 lg copies/μL.The recovery rate of samples was between 95% and 105%.Results showed that the content of T.vulgaris fluctuated and reached a maximum of (6.06±0.02) lg copies/g on the 5th day.The content of T.intermedius increased first and then decreased, and reached the highest on day 7, which was (6.33±0.13) lg copies/g.The content of T.daqus fluctuated from day 0 to day 12, decreased from day 12 to day 14, and then continued to increase, reaching a maximum of (7.62±0.02) lg copies/g at the end of fermentation.The RT-qPCR method established in this study can be used for specific identification and rapid quantification of three Thermoactinomyces species during the fermentation process of Daqu, which provides a method for further explaining the function of Thermoactinomyces in the brewing process of Baijiu.
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