关键词: 转基因, 抗逆大豆IND-ØØ41Ø-5, 转化体特异性, 实时荧光定量PCR

Abstract:

【Objective】Stress-resistant soybean IND-ØØ41Ø-5 has been authorized as a safety certificate for imported processed raw materials in China. This study aims to develop a specific, accurate, and sensitive real-time quantitative PCR (qPCR) assay for the quantification of the stress-resistant soybean IND-ØØ41Ø-5 event, providing a precise measurement technique for regulating its safety in China. 【Method】18 pairs of primers and probes were designed using Beacon designer 8.0 software for the 5′ end flanking sequence of the stress-resistant soybean IND-ØØ41Ø-5 event, in combination with one pair of primer and probe providing by Instituto de Agrobiotecnologia Rosario (INDEAR) S.A. Subsequently, the specific screening of the primers and probes was performed using real-time PCR technology, and one pair of candidate primer and probe was selected. The reaction system parameters, such as primer and probe concentration, were optimized during the establishing the qPCR method. A specificity test was performed using different test samples. The pure stress-resistant soybean IND-ØØ41Ø-5 genomic DNA was serially diluted into standard solution templates, and standard curves were plotted to investigate the linear dynamic range of the qPCR method. Test samples with copy number ratios of 5%, 1% and 0.1% were prepared by mixing IND-ØØ41Ø-5 genomic DNA with non-GM counterpart to evaluate the accuracy of the qPCR method. The limit of detection (LOD) was detected by using test samples with copy number ratios of 0.05% and 0.025%. The limit of quantification (LOQ) was determined after 16 tests on samples with a copy number ratio of 0.1%. Finally, the technical parameters of qPCR assay for the stress-resistant soybean IND-ØØ41Ø-5 event were determined. The specificity, LOD, LOQ and accuracy of the qPCR method were validated by eight qualified testing laboratories. The repeatability and reproducibility of the qPCR were evaluated by Cochran′s test and Grubbs' test, and the measurement uncertainty of the accuracy samples were pre-evaluated by linear least-square method. 【Result】The RBORD-F1/RBORD-R1/RBORD-P1 primer and probe combination was selected as a candidate to establish the qPCR for the stress-resistant soybean IND-ØØ41Ø-5 event, with an amplified fragment of 138 bp. The optimized reaction system had a final concentration of 0.4 μmol·L^-1 for the primer and 0.2 μmol·L^-1 for the probe. Standard curves of IND-ØØ41Ø-5 and Lectin gene assay showed good linearity with the dynamic range from 33 to 83190 copies of genomic DNA. The qPCR can accurately quantify 5%, 1%, and 0.1% content of IND-ØØ41Ø-5 test samples with less than 25% bias and relative standard deviation (RSD). The LOD was determined to be 0.05%, and the LOQ was 0.1%. After validation by eight qualified laboratories, the results indicated that the method was stable, specific and had good repeatability and reproducibility, with the LOD of 0.05% and LOQ of 0.1%. After pre-evaluating the measurement uncertainty, the content of IND-ØØ41Ø-5 in the five test samples was found to be (0.10±0.02)%, (0.53±0.09)%, (1.05±0.18)%, (2.05±0.34)% and (5.18±0.87)%, respectively. These results demonstrate the accuracy and reliability of the qPCR method established in this study for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event components. 【Conclusion】This study successfully developed a specific, accurate, and sensitive qPCR assay for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event using real-time PCR technology. The results show that method is capable of achieving precise measurement and reliable quantification of IND-ØØ41Ø-5 event components.

Key words: transgenic, stress-resistant soybean IND-ØØ41Ø-5, event-specific, real-time quantitative PCR (qPCR)