转基因苜蓿草J163品系特异性实时荧光PCR检测方法的建立

Establishing an event-specific real-time polymerase chain reaction detection method for genetically modified alfalfa events J163

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 real-time PCR detection method for genetically modified (GM) Roundup Ready alfalfa events J163, which has not been authorized by the Ministry of Agriculture of China. Method The specific primer pairs and probe based on the 5’junction sequence spanning the alfalfa DNA and inserted P-eFMV fragment of J163 were designed and then the real-time PCR detection system was established. The specificity, sensitivity and repeatability were analyzed. Results The real-time PCR method was specific for GM alfalfa J163 detection, the limit of detection(LOD) were 15 pg J163 genomic DNA or 10 copies of alfalfa J163 haploid genomic DNA. Repeatability of the established event-specific real-time PCR method for GM alfalfa J163 was assessed and the standard deviation (SD) and the relative standard deviation (RSD) were all in the acceptable range. Conclusion The established event-specific quantitative real-time PCR method for GM alfalfa J163 detection has a high specificity and a good sensitivity, and is suitable for quantification of J163 samples quickly and accurately.