Development, Optimization, and Single Laboratory Validation of an Event-Specific Real-Time PCR Method for the Detection and Quantification of Golden Rice 2 Using a Novel Taxon-Specific Assay

Abstract

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

Applicability of quantitative polymerase chain reaction (qPCR) assays for common bean authentication in processed food

“…FAS assay presented an early Cq compared to LEC and FEI assay. The Cq values variability range of an endogenous reference could not be >1 (Jacchia et al ., ). A novel taxon‐specific assay was developed for rice, majority of rice varieties presented similar Cq values, but some rice varieties were considered outliers with lower Cq values (Jacchia et al ., ).…”

“…The Cq values variability range of an endogenous reference could not be >1 (Jacchia et al ., ). A novel taxon‐specific assay was developed for rice, majority of rice varieties presented similar Cq values, but some rice varieties were considered outliers with lower Cq values (Jacchia et al ., ). A new endogenous reference was developed for sugar beet (Chaouachi et al ., ), stable similar average Cq values were observed for majority of varieties, although variable Cq values were observed for triploid varieties and polyploid fodder beet.…”

“…The LEC assay presented efficiency values ranging from 94% to 101% and R 2 was 0.99, LOD was ten DNA copies, with a mean Cq of 33.5. Regarding efficiency values in the range 90–110% and LOD around 100 copy number, FAS assay are in accordance with FEI and LEC assays and with other endogenous reference assays developed for sugarbeet (Chaouachi et al ., ), rice (Jacchia et al ., ) and rapeseed (Wu et al ., ).…”

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

“…When the concentration of Taq was doubled for the secondary PCR, all three amplicons were clearly visualized. This suggests that the catalytic action of Taq is a rate-limiting factor in achieving simultaneous products of multiple templates in the same reaction mixture (35).…”

In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

“…The absence of copy number variation within the taxon is generally verified on a representative collection of cultivars, by using real-time PCR (Rønning et al 2006;Vautrin and Zhang 2007;Chaouachi et al 2008;Jacchia et al 2015). The range of variability of Cq values in amplification should not exceed 1 Cq within the taxon (European Network of GMO Laboratories (ENGL) 2015).…”