Due to the increasing number of authorized events in the European Union, it is crucial for the official laboratories to enforce market control to detect and quantify genetically modified organisms. In this study, an in-house validation of quantitative duplex ddPCR methods was performed involving MON87701, MON87769, MON89788 and CV-127-9 assays with respect to the lectin reference gene. Since the ddPCR methods provide accurate quantification, show less sensitivity to PCR inhibitors and are more suitable for multiplexing compared to the real-time PCR, the optimization of the existing assays was performed with the exception of MON87701, according to the JRC Guidance documents and technical reports. However, some concerns related to practical settings for the quantitative multiplex of ddPCR methods and their validation were encountered; therefore, a general workflow to develop and validate a ddPCR-based method is shown. The obtained data and the validation performance parameters such as specificity, cross-talk, robustness, dynamic range, linearity, the limit of quantification, trueness and precision comply with international recommendations for GMO quantification methods. The duplex ddPCR methods here investigated are equivalent in terms of performance compared to the singleplex real-time PCR methods, showing higher flexibility and cost effectiveness.

In the European Union, the food and feed containing more than 0.9% of approved genetically modified organisms (GMOs) per ingredient must be labeled before placed on the market. In this legislative context, the official control laboratories have to perform validated PCR assays, according to the principles and requirements of ISO/IEC 17025 standard, regarding event-specific methods for the detection, identification and quantification of GMOs. In recent years, with the advent of digital PCR (dPCR) techniques, a growing number of laboratories have transferred the previously validated real-time PCR testings into a dPCR format. Compared to real-time PCR, the dPCR offers the advantage to provide accurate quantification without the need for external calibration samples, show less sensitivity to PCR inhibitors and is more suitable for multiplexing. In this study, an in-house validation of quantitative duplex dPCR methods was performed involving MON-04032-6 and MON89788 assays with the lectin reference gene, on the two different platforms Bio-Rad QX200 and Qiagen QIAcuity. All evaluated data and the validation parameters agree with the acceptance criteria validation performance parameters according to the JRC Guidance documents and technical reports in both platforms. The duplex PCR methods here investigated are equivalent in terms of performance to the singleplex real-time PCR method and suitable to perform a collaborative trial for a full validation.

In recent years, CRISPR-Cas9 technology has become a powerful and indispensable tool for targeted mutagenesis in plants, including applications such as gene knockout, prime-editing, multiplex gene editing, and regulation of gene transcription. As the number of potential genome editing approaches expands at a very fast pace, rapid, efficient, and cost-effective analytical strategies are needed to screen large numbers of mutants, including the detection of off-target events. In this study, we reported a detection method based on High-Resolution Melting (HRM) analysis to discriminate between wild-type (wt) and edited lines of different varieties of Vitis vinifera and grapevine rootstocks. Those edited lines were obtained through Agrobacterium tumefaciens mediated transformation of embryogenic calli using the CRISPR/SpCas9 system and targeting VviEPFL9-1 and VviEPFL9-2, two paralogous genes involved in stomata cell fate induction. The method clearly distinguished between the wt allele and the mutated one and was partially effective in distinguishing different types of mutation. Moreover, HRM data elaboration based on a Principal Component Analysis (PCA) allowed one to group populations of lines which originated from the same transformation event. Our study demonstrates the reliability of HRM as a fast and cost-effective diagnostic tool for the screening of edited lines and the evaluation of off-target events.

In the European Union, mandatory labeling of food and feed products is required when authorized genetically modified organisms (GMOs) exceed 0.9% per ingredient, necessitating reliable analytical methods for official control laboratories. Event-specific PCR assays validated according to ISO/IEC 17025 are the reference approach for GMO detection, identification, and quantification. The growing use of digital PCR (dPCR) has encouraged the adaptation of real-time PCR methods to dPCR-based strategies, as dPCR enables absolute quantification without calibration standards, shows reduced sensitivity to inhibitors, and allows for the design of a multiplex assay. In this study, an in-house validation of a duplex dPCR assay targeting the maize GM event NK603 and the HMG reference gene was performed on three platforms: Bio-Rad QX200™ (Pleasanton, CA, USA), Qiagen QIAcuity (Venlo, The Netherlands), and Thermo Fisher QuantStudio Absolute Q (Waltham, MA, USA). All validation parameters met the Joint Research Centre (JRC) acceptance criteria. In particular, this assay demonstrated high specificity, sensitivity (limit of quantification or LOQ < 35 copies per reaction), precision, and trueness (RSDr and bias <25%). The data indicate that the duplex dPCR assay can be used for routine GMO analysis and future collaborative validation studies.