摘要
建立多重串联式PCR(MT-PCR)的基因碟片技术用于转基因大豆GTS40-3-2的检测。针对GTS 40-3-2的常见外源基因NOS终止子、CP4-EPSPS、Ca MV35S启动子和大豆内源基因Lectin设计引物,同时针对外源基因插入位点的旁临序列设计品系特异性引物。首先进行一次循环数较少(15 cycles),引物浓度较低(0.1μmol/L)的高通量多重PCR,以均匀地扩增各基因模板,同时避免引物之间的竞争,然后利用巢式荧光定量PCR检测各个基因。根据熔融曲线分析结果,灵敏度高于普通荧光定量PCR法1个数量级。该方法能够快速、高通量、准确地检测转基因大豆GTS40-3-2中的多种转基因成分,并能对该品系进行分析,重复性好,适合转基因的高通量、定量检测,可用于特异性检测转基因大豆GTS40-3-2,具有较好的应用价值。
This study has developed a multiplex tandem polymerase chain reaction(MT-PCR) gene disk assay for the detection of genetically modified soybean GTS 40-3-2. The MT-PCR based on the detection of NOS terminator,Ca MV35 S promoter, CP4-EPSPS gene, soybean endogenous gene Lectin and event-specific primers of genetically modified soybean GTS 40-3-2. The MT-PCR composed of two PCR rounds: using a small number of cycles(15 cycles) for high-throughput multiplex amplification at first round to avoid competition between amplicons; and then, second round real-time PCR was used to detect individual genes which can be confirmed by melting curve analysis. The MT-PCR assay was rapid(<2 h), high-throughput(5 genes of each sample), sensitive(0.001 ng/μL), and specific in detecting GM soybean GTS 40-3-2. The MT-PCR assay is high-throughput, rapid, and can be used for specific validation of transformation event in transgenic soybean GTS40-3-2.
引文
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