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In this study, the specific fragment of turkey chromosome Z-DNA sequence was selected as the target sequence for turkey-derived material detection. A real-time polymerase chain reaction (real-time PCR) method was established, which had good interspecies specificity and interspecies consistency. The target sequence was cloned into the plasmid pUC57, and after being diluted to different concentrations, the plasmid was tested by real-time PCR. The absolute detection limit of this method was 5 copies/PCR reaction. An international collaborative validation trial was conducted to validate this method. The results showed that the false positive and negative rates of the proposed method were both 0%, and the absolute detection limit was 5 copies/PCR reaction. The results of qualitative analysis of the diluted plasmid showed that the inter-laboratory standard deviation was 0.30, less than the maximum permitted value of 1; at a detection probability of 95%, the absolute detection limit was 3.2 copies/PCR reaction, less than the maximum allowable value of 20 copies/PCR reaction. Based on voting results and reviewers’ comments, this method was approved by the International Organization for Standardization (ISO) as an international standard method (ISO/TS 20224-8:2022).

Background: To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed., Results: To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5'-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients., Conclusions: The developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products.

The qualitative event-specific polymerase chain reaction detection method of genetically modified (GM) RT73 rapeseed was developed based on the cloned 3' end flanking sequence of RT73 rapeseed integration. The specificity of the method for GM RT73 rapeseed was validated using several different GM rapeseed lines, GM maize lines, GM soybean line, non-GM rapeseed, and other non-GM crops. In this study, the developed method was validated through an interlaboratory study by 12 laboratories from 6 countries. The sensitivity of this method was evaluated using several mixed rapeseed meals with different GM RT73 rapeseed contents from 5.0 to 0.01% prepared by our laboratory. The evaluated results showed that all of the rapeseed endogenous reference high mobility group protein gene (HMG I/Y), figwort mosaic virus 35S (FMV 35S) promoter, and RT73 event-specific fragment could be detected from rapeseed samples at 0.1% (w/w) with a confidence level of more than 95%. All results from the 12 laboratories indicated that the developed method could be considered fit for the detection and identification of GM RT73 rapeseed.