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目的建立针对我国农业部未颁发农业转基因生物安全证书的转基因苜蓿草品系J163品系特异性实时荧光(Polymerase Chain Reaction,PCR)检测方法。方法利用Taq Man实时荧光PCR(real-time PCR)技术,根据转基因苜蓿草品系J163 5’端外源插入片段P-e FMV与苜蓿草基因组DNA之间的邻接区序列设计引物和探针,建立了转基因苜蓿草J163品系特异性实时荧光PCR检测方法,并对本方法的特异性、灵敏度及可重复性进行了测定。结果建立的检测方法特异于转基因苜蓿草J163成分检测,检测最低DNA浓度为(1imit of detection,LOD)为15 pg,相当于9拷贝转基因苜蓿草J163基因组DNA,重复性试验显示,其标准偏差(Standard deviation,SD)和相对标准偏差(relative standard deviation,RSD)均在可接受范围内。结论本研究建立的转基因苜蓿草J163品系特异性实时荧光PCR检测方法特异性好,灵敏度高,能够快速、准确、稳定地对转基因苜蓿草J163成分进行检测分析。
Objective To establish a specific real-time fluorescence (Polymerase Chain Reaction, PCR) method for detecting J163 transgenic alfalfa lines that have not been issued with the certificate of agricultural genetically modified organisms by Ministry of Agriculture of China. Methods TaqMan real-time PCR (real-time PCR) was used to design primers and probes according to the sequence of the adjacent region between the exogenous DNA fragment of Pe FMV and alfalfa at the 5 ’end of the transgenic alfalfa grass line J163. Alfalfa J163 strain specific real-time fluorescence PCR detection method, and the specificity, sensitivity and repeatability of the method were determined. Results The detection method was specific to the detection of transgenic alfalfa J163. The minimum detectable DNA concentration (LOD) was 15 pg, which was equivalent to 9 copies of transgenic alfalfa J163 genomic DNA. Repeatability tests showed that the standard deviation ( Standard deviation, SD) and relative standard deviation (RSD) were within acceptable range. Conclusion The specific real-time fluorescent PCR detection method of transgenic alfalfa J163 strain established in this study has good specificity and high sensitivity, and can detect and analyze the composition of transgenic alfalfa J163 rapidly, accurately and stably.