多重串联式PCR基因碟片技术检测转基因大豆GTS 40-3-2
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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.
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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[14]魏霜,陈贞,芦春斌,等.多重PCR检测转基因水稻的转基因成分[J].食品科学,2012,33(12):159-162.
[15]魏霜,陈贞,马骏,等.基于多重串联式PCR的基因碟片技术检测转基因作物[J].中国农业科学,2013,45(5):1010-1016.
[2]LAM W Y,YEUNG A C M,TANGJ W,et al.Rapid Multiplex nested PCR for detection of respiratory viruses[J].Journal of Clinical Microbiology,2007,45(11):3631-3640.
[3]SCHMIDT A,SAHOTA R,POPE D S,et al.Detection of genetically modified canola using multiplex PCR coupled with oligo nucleotide microarray hybridization[J].Journal of Agricultural and Food Chemistry,2008,56(16):6791-6800.
[4]ZEITLER R,PIETSCH K,WAIBLINGER H U.Validation of real-time PCR methods for the quantification of transgenic contaminations in rape seed[J].European Food Research and Technology,2002,214(4):346-351.
[5]王永,兰青阔,赵新,等.转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用[J].中国农业科学,2009,42(4):1473-1477.
[6]STANLEY K K.Multiplexed tandem PCR:Gene profiling from small amounts of RNA using SYBR Green detection[J].Nucleic Acids Research,2005,33(20):e180-e180.
[7]LAU A,SORRELL T C,CHEN S,et al.Multiplex Tandem PCR:A novel platform for rapid detection and iden tification of fungal pathogens from blood culture specimens[J].Journal of Clinical Microbiology,2008,46(9):3021-3027.
[8]LAU A,SORRELL T C,LEE O C,et al.Colony multiplex tandem PCR for rapid,accurate identification of fungal cultures[J].Journal of Clinical Microbiology,2008,46(12):4058-4060.
[9]STARK D,AL-QASSAB S E,BARRATT J L N,et al.Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp.,Dientamoeba fragilis,Entamoeba histolytica,and Giardia intestinalis in clinical stool samples[J].Journal of Clinical Microbiology,2010,49(1):257-262.
[10]SZEWCZUK E,THAPA K,ANNINOS T,et al.Rapid semi-automated quantitative multiplex tandem PCR(MTPCR)assays for the differential diagnosis of influenza-like illness[J].BMC Infectious Diseases,2010,10(1):113.
[11]JAMES C.2014年全球生物技术/转基因作物商业化发展态势[J].中国生物工程杂志,2015,35(1):1-14.
[12]DONG W,YANG L,SHEN K,et al.GMDD:a database of GMO detection methods[J].BMC bioinformatics,2008,9(1):260.
[13]KIM J,JEONG D,KIM Y,et al.Development of a multiplex PCR method for testing six GM soybean events[J].Food Control,2013,31(2):366-371.
[14]魏霜,陈贞,芦春斌,等.多重PCR检测转基因水稻的转基因成分[J].食品科学,2012,33(12):159-162.
[15]魏霜,陈贞,马骏,等.基于多重串联式PCR的基因碟片技术检测转基因作物[J].中国农业科学,2013,45(5):1010-1016.