Abstract
Owing to the economic value of its foliage, tobacco (Nicotiana tabacum) is cultivated all across the world. For the detection of genetically modified (GM) tobacco, there is a lack of universal standard material which ultimately limits the detection methods because the accuracy and comparability of the results cannot be ensured. Here, we prepared a reference plasmid “pGMT27” for the detection of GM tobacco, which was 18,296 bp in length harboring two of the tobacco endogenous and seven exogenous genes. By using qualitative PCR test for the nine genes, 10 copies were used for plasmid sensitivity. In the quantitative real-time PCR (qPCR) assays with pGMT27 as a calibrator, the reaction efficiencies for P-35S and NR were 101.427% and 98.036%, respectively, whereas the limit of detection (LOD) and limit of quantification (LOQ) were 5 copies and 10 copies per reaction. For standard deviation (SD) and relative standard deviation (RSD) of the Ct values, the repeatability values were from 0.04 to 0.42 and from 0.18% to 1.29%, respectively; and the reproducibility values were from 0.04 to 0.39 and from 0.18% to 1.14%, respectively. For the unknown sample test, the average conversion factor (Cf) was 0.39, and the accuracy bias was from −15.55% to 1.93%; for precision, the SD values ranged from 0.02 to 0.62, while RSD values were from 1.34% to 10.6%. We concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.
Highlights
Tobacco is one of the most widely cultivated nonfood crops and has emerged as an extensively investigated model plant with the accelerated development of molecular biology research
For testing sensitivity in the qualitative PCR, nine targets by using 106, 105, 104, 103, 102, 50, 10, 5, and 1 copies of the pGMT27 plasmid as templates were measured by the limit of detection (LOD) method
Nine targets were detected as low as 5 copies, which meant that the lowest testing level was 5 copies (Figure 2)
Summary
Tobacco is one of the most widely cultivated nonfood crops and has emerged as an extensively investigated model plant with the accelerated development of molecular biology research. Attributable to its agronomic importance, tobacco is grown in over 125 countries for its foliage, mainly consumed as cigarettes, cigars, and snuff. When entering into the market, it is indispensable for the tobacco companies to enhance controls on import and export of the tobacco product. In many countries, respecting public’s right to information and education, the GMO threshold levels of food and feed products have already been established [6,7,8], to ensure the accurate and comparable measurements, selection of standard material for GMO detection becomes critical [9]. E standard reference material includes matrix and plasmid. Some plasmids as calibrators have been used for transgenic soybean, maize, cotton, and other crops, including ERM-AD413, ERM-AD415, ERM-AD427, and ERM-AD425. Some plasmids as calibrators have been used for transgenic soybean, maize, cotton, and other crops, including ERM-AD413, ERM-AD415, ERM-AD427, and ERM-AD425. ey are used to calibrate the transgenic maize MON810, NK603, 98140, and transgenic soybean 356043 [11]
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