酵母菌mRNA提取技术及实时荧光定量逆转录聚合酶链式反应检测方法研究

Study on the extraction of yeast mRNA and the detection method of reverse transcription real-time fluorescence quantitative polymerase chain reaction

中文关键词:  酵母菌  mRNA提取  实时荧光定量逆转录聚合酶链式反应  几丁质合成酶基因2

英文关键词:yeasts  mRNA extraction  reverse transcription real-time fluorescence quantitative polymerase chain reaction  chitin synthase 2

中文摘要:

      目的 探究一种高效的酵母菌信使核糖核酸(messenger RNA, mRNA)提取方法, 并建立相应的酵母菌基因实时荧光定量逆转录聚合酶链式反应(reverse transcription real-time fluorescence quantitative polymerase chain reaction, RT-qPCR)特异检测方法。方法 采用自主研发的高性能二氧化硅磁珠为mRNA捕获载体, 通过链霉亲和素磁珠(SM)-Oligo(dT)联合Trizol裂解捕获的方法, 优化磁珠浓度、磁吸附时间等, 建立高效的酵母菌mRNA提取技术。根据酵母菌几丁质合成酶基因2 (chitin synthase 2, CHS2)设计引物, 建立一种酵母菌特异性的RT-qPCR检测方法, 对该方法的特异性、稳定性进行验证。结果 在优化提取条件下, 本研究可以实现不同浓度酵母菌的mRNA提取, 低浓度菌液(2×103 cfu/mL)也能提取2.13 μg mRNA。本研究建立的酵母菌检测方法检出线性范围为2×103~2×107 cfu/mL, 线性方程Y=–1.69X+33.412 (r2=0.9966), 该方法对目标酵母菌表现出高度的特异性。结论 本研究建立了酵母菌特异性磁珠法mRNA提取技术及RT-qPCR检测方法, 该方法有较好的特异性、灵敏度, 为食品及科研中酵母菌的定量检测提供新技术。

英文摘要:

      Objective To explore an efficient method for extracting messenger RNA (mRNA) from yeast and establish a specific detection method by reverse transcription real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Methods A high-performance silica magnetic beads by self-developed were used as carrier, an efficient yeast mRNA extraction technique was developed by combining streptavidin magnetic beads (SM) conjugated with Oligo(dT) and Trizol lysis, followed by optimization of magnetic bead concentration and magnetic adsorption time. Primers based on the yeast chitin synthase 2 (CHS2) gene, established a yeast-specific RT-qPCR assay, and its specificity and stability were validated.?Results Under optimized extraction conditions, the method enabled extraction yeast mRNA from various concentration, yielded 2.13 μg mRNA even from a low-concentration (2×103 cfu/mL). This yeast detection methodexhibited a wide linear range from 2×103 to 2×107 cfu/mL, with a linear equation of Y=–1.69X+33.412 (r2=0.9966), the method exhibited high specificity for the target yeast.?Conclusion This study successfully establish a mRNA extraction technique by yeast-specific magnetic bead-based and a RT-qPCR detection method, exhibits good specificity and sensitivity, provide a new technology for the detection of yeast contamination in food and scientific research.