术语新基因组技术(NGT)是一个总称,用于描述各种技术,这些技术可以改变生物体的遗传物质,并且自2001年现有的转基因生物(GMO)立法以来已经出现或已经开发通过。用于检测欧洲转基因生物的分析框架是一个既定的单一协调程序,对于转基因食品和饲料的授权是强制性的,从而产生一个可靠的,透明,和有效的GMO产品标签计划。然而,NGT产品可以挑战欧盟现行监管体系的实施和执行,特别涉及检测不含外来遗传物质的NGT产品。因此,当前的检测方法可能无法满足最低性能要求。尽管现有的检测方法可能能够检测和量化基因组中的微小变化,这并不一定确认受GMO法规约束的NGT产品与其他产品之间的区别。因此,本研究通过测试当前基因组数据库中扩增子和引物集搜索的生物信息学管道,为PCR系统特异性的计算机模拟预测提供了一种逐步的方法.此外,它还根据经验测试了在计算机分析期间评估的PCR系统。CRISPR-Cas9在拟南芥中产生的两种突变基因型被用作案例研究。总的来说,我们的结果表明,为识别grf1-3基因型中的核苷酸插入而开发的单个PCR系统在数据库中具有多个匹配,这不能区分这种突变事件。经验分析进一步支持这种证明。相比之下,第二个突变的基因型,grf8-61,包含一个-3bp的缺失,在序列变体数据库中未产生任何匹配。然而,在经验测定期间引物序列效率不高。我们的方法代表了NGT检测分析方法决策的第一步,identification,并根据欧洲标签法规进行量化。
The term new genomic techniques (NGTs) is an umbrella term used to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework used to detect GMOs in Europe is an established single harmonized procedure that is mandatory for the authorization of GM food and feed, thus generating a reliable, transparent, and effective labeling scheme for GMO products. However, NGT products can challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material. Consequently, the current detection methods might fail to meet the minimum performance requirements. Although existing detection methods may be able to detect and quantify even small alterations in the genome, this does not necessarily confirm the distinction between products resulting from NGTs subject to the GMO legislation and other products. Therefore, this study provides a stepwise approach for the in silico prediction of PCR systems\' specificity by testing a bioinformatics pipeline for amplicon and primer set searches in current genomic databases. In addition, it also empirically tested the PCR system evaluated during the in silico analysis. Two mutant genotypes produced by CRISPR-Cas9 in Arabidopsis thaliana were used as a case study. Overall, our results demonstrate that the single PCR system developed for identifying a nucleotide insertion in the grf1-3 genotype has multiple matches in the databases, which do not enable the discrimination of this mutated event. Empirical assays further support this demonstration. In contrast, the second mutated genotype, grf8-61, which contains a -3 bp deletion, did not yield any matches in the sequence variant database. However, the primer sequences were not efficient during the empirical assay. Our approach represents a first step in decision making for analytical methods for NGT detection, identification, and quantification in light of the European labeling regulations.