Ivane Beritashvili Center of Experimental Biomedicine, 14 Gotua str., Tbilisi 0160, Georgia.
School of Natural Sciences and Medicine, Ilia State University, 3/5 Kakutsa Cholokashvili Ave, Tbilisi 0162, Georgia.
Biosensors (Basel). 2021 Nov 26;11(12):481. doi: 10.3390/bios11120481.
Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.
转基因植物和食品的变应原性评估对于食品安全、标签法规和健康保护非常重要。本研究的目的是开发一种用于转基因大豆评估的有效多过敏原诊断方法。为此，采用了多重聚合酶链反应（PCR）与 DNA 芯片技术相结合的方法。本研究集中在抗除草剂 Roundup Ready 大豆（RRS）上，使用了一组由 0、0.1%、0.5%和 10%RRS 的认证参考材料。从技术上讲，该程序包括设计 PCR 引物和探针；基因组 DNA 提取；单重和多重 PCR 系统的开发；琼脂糖凝胶电泳的 DNA 分析；微阵列的开发、杂交和扫描。不对称多重 PCR 方法的使用显示出与生物芯片探针进行 DNA 杂交的非常有效。我们证明，新开发的四种 PCR 方法与 DNA 芯片相结合，能够同时鉴定三种主要的内源性过敏原，即 Gly m Bd 28K、Gly m Bd 30K 和凝集素，以及在抗除草剂 Roundup Ready GMO 中表达的 5-烯醇丙酮酸莽草酸磷酸合酶（epsps）。本研究中开发的方法可用于准确、廉价和快速检测食物过敏原。
