Federal Research Centre of Nutrition, Biotechnology, and Food Safety, Moscow, Russia.
Bull Exp Biol Med. 2023 Jul;175(3):393-398. doi: 10.1007/s10517-023-05874-3. Epub 2023 Aug 10.
The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.
该研究证实了有必要实施遗传修饰微生物（GMM）生物安全性的分子遗传评估算法，并开发标准化方法来测试产生酶、生物活性物质和其他微生物合成产物的遗传修饰菌株，然后再将其用于食品工业。对微生物生产者及其相关食品产品进行 GMM 相关 DNA 分析，结果表明，在酶制剂和工业遗传修饰曲霉菌属生产者的菌丝体中，标记基因 amp 和 lacZ 的发生率很高。通过包括提取物额外纯化阶段、使用通用 ITS 引物进行 PCR 评估其纯度以及在进行分子遗传分析之前确定样品中有效 DNA 浓度，优化了从霉菌真菌菌丝体中提取 DNA 的程序。为了鉴定霉菌真菌（酶制剂的生物技术生产者）和进行基因分型，我们对 Sanger 测序方法进行了适应性改造。使用这种改良的方法，我们确定了五种难以鉴定的曲霉菌属菌株的种属。为了鉴定子囊菌门的密切相关的微型真菌，我们开发了一种基于扩增总 DNA 并用扩展引物组进行 DNA 测序的毛细管电泳的基因分型算法。
