荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆

目的：建立实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测转基因DAS-44406-6品系大豆的定性检测方法和使用数字PCR检测转基因DAS-44406-6品系大豆的定量检测方法。方法：针对转基因DAS-44406-6大豆品系,进行5’-RACE,测定该品系转基因大豆外源片段与大豆染色体重组的边界序列,并根据该边界序列设计引物和探针。使用23 种非DAS-44406-6品系转基因植物作为阴性对照测试实时荧光PCR引物和探针的特异性,以DAS-44406-6品系样品制备6 个含量梯度的样品进行检测低限实验。使用数字PCR技术进行定量检测,并确定定量检测的低限。结果：建立的转基因DAS-44406-6大豆品系的实时荧光PCR特异性检测方法品系鉴定特异性较强,实时荧光PCR检测方法的检测低限在模板DNA浓度为100 ng/反应时,为0.01%的转基因大豆含量,约为16.6 个拷贝的DAS-44406-6基因组DNA;数字PCR检测方法的检测低限在模板DNA浓度为0.5 ng/反应、转基因大豆含量为1%时,相对标准偏差为0.7%。因此,建立的转基因DAS-44406-6大豆品系实时荧光PCR和数字PCR特异性检测方法符合转基因检测的要求。     

转基因玉米GA21品系LAMP检测引物的筛选及方法的建立

根据转基因玉米GA21品系的特异序列设计了3套环介导等温扩增(LAMP)引物。通过反应温度优化、特异性测定,筛选到1套特异性好的LAMP引物,包括内引物、外引物和环引物各1对。在LAMP反应体系中加入SYBR Green I荧光染料,在实时荧光定量PCR仪上进行实时监测,对筛选到的LAMP引物进行反应条件、反应体系的优化,建立了转基因玉米GA21品系LAMP检测方法。对该方法的特异性、灵敏度、稳定性进行评价,结果表明建立的LAMP检测方法能特异、灵敏、稳定地检测转基因玉米GA21品系,检测限达到0.05%。将建立的LAMP检测方法应用于玉米及其制品中GA21品系的检测,检测结果与实时荧光PCR法的结果完全一致。     

多重串联式PCR基因碟片技术检测转基因大豆GTS 40-3-2

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。     

转基因大豆MON89788实时荧光PCR检测方法的建立

为实现转基因大豆MON89788的标识管理,针对转基因大豆MON89788的品系特异性序列设计引物和TaqMan探针,建立转基因大豆MON89788实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法,并对该方法的特异性、灵敏度和重复性进行检测。结果显示：建立的转基因大豆MON89788实时荧光PCR检测方法能扩增出127 bp的产物,特异性强,灵敏度达到0.1%,约为40 个单倍体基因组拷贝,检测重复性好,可成功应用于实际样品检测。因此,建立的转基因大豆MON89788实时荧光PCR检测方法可以应用于转基因大豆MON89788大豆及其制品的检测。     

转基因抗除草剂玉米DAS-40278-9品系特异实时荧光定量PCR检测方法的建立

根据转基因抗除草剂玉米DAS-40278-9外源基因的旁侧序列,采用ABI Primer Express 3.0设计引物和Taq Man探针,建立转基因抗除草剂玉米DAS-40278-9品系特异实时荧光定量PCR检测方法。采用该检测方法对DAS-40278-9玉米含量为1%(质量分数)的标准样品进行检测,结果显示,采用建立的方法获得的标准曲线,通过斜率、相关系数、扩增效率判断是可靠的,待测样品的定量检测结果为1.024%,非常接近真实值。本试验表明建立的转基因抗除草剂玉米DAS-40278-9品系特异定量PCR检测方法的特异性好,灵敏度和准确度高,值得推广应用。     

北极苹果结构特异性实时荧光聚合酶链式反应检测方法建立

目的为实现转基因北极苹果(Arctic~(TM) apple)目的标识管理,建立特异性实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法。方法针对转基因北极苹果特异性序列设计引物和TaqMan探针,建立转基因北极苹果实时荧光PCR检测方法,并对该方法的特异性、灵敏度和重复性进行检测。结果建立的转基因北极苹果实时荧光PCR特异性强,定量检测限为20拷贝,扩增效率为96%,检测重复性良好。结论建立的特异性实时荧光PCR法可应用于转基因北极苹果的鉴定。     

实时荧光定量PCR技术检测转基因大豆方法的建立

采用实时荧光定量PCR技术 ,通过使用特异的引物和探针 ,对大豆中的内源基因Lectin和转基因大豆中的外源基因EPSPS进行了定量检测 ,建立了Monsanto公司生产的商业化转基因大豆Roundup Ready○R的定量PCR检测方法。该方法的检测灵敏度 <0 0 1% ,是国际上设定的转基因最低限量的 10 0倍     

SYBR Green实时荧光定量PCR检测大豆转基因成分

采用SYBR Green实时荧光定量PCR技术,建立了食品中大豆转基因成分的定量检测方法。通过设计特异引物,扩增内源参照基因lectin和转基因靶基因CP4EPSPS,建立两种基因的拷贝数-CT标准曲线,根据标准曲线方程计算样品中的转基因含量,并且通过熔解曲线分析扩增反应特异性。结果表明,lectin和CP4EPSPS基因标准曲线线性关系好,R2值分别为0.9984和0.9953,方法的回收率为95%～110%,检测限为0.01%。本检测方法具有快速、灵敏、准确、特异、高通量等优点,可以作为食品中大豆转基因成分的定量检测方法。     

转基因苜蓿草J163品系特异性实时荧光PCR检测方法的建立

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。     

实时荧光PCR方法检测转基因豆粕的研究

以美国、阿根廷和转基因大豆标准品为材料,利用设计的特异性引物和探针,建立了实时荧光定量PCR技术检测抗草甘膦转基因豆粕的方法,成功检测出美国和阿根廷抗草甘膦转基因豆粕的内源参照基因lectin、CaMV35S/CTP of EPSPS边界序列和NOS终止子,确定了实时荧光PCR方法检测抗草甘膦转基因豆粕的灵敏度为0.1%。     

An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin

Transgenic rice 114-7-2 is a newly developed transgenic rice line of producing human serum albumin (HSA). It has attracted much attention because of its economic potential. This paper was designated to discover the integration site of the transgenic HSA rice line 114-7-2 and to establish event-specific methods for qualitative and quantitative detection of the transgenic HSA rice based on the border junction fragment. One gene fragment of 5′ flanking region was successfully isolated using the TAIL-PCR methods. The fragment sequence showed that a 454-bp junction fragment contained 75 bp of T-DNA sequence and 379 bp of rice genome DNA, which is located in chromosome 4. Event-specific real-time PCR method for HSA rice line 114-7-2 was established with the primers (HSA-F/HSA-R) and the probe (HSA-P) targeting the 454-bp junction region. The qualitative PCR assay showed the limit of detection was 0.01 %. In the event-specific quantitative detection method, the LOQ for 114-7-2 HSA rice was estimated to be 0.025 ng or 50 copies. The method developed in this study is highly specific, sensitive, and reliable for transgenic HSA rice sample detection.     

Event-Specific Qualitative and Quantitative Detection in Transgenic Soybean OsDREB3 Based on the 5′ Flanking Sequence

With the development of genetically modified organisms, labeling regulations have been introduced that require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-the art. Using adaptor PCR, we analyzed the flanking sequences of exogenous integrant in transgenic soybean OsDREB3, which has resistance genes. In this study 5′ region flanking sequences of exogenous gene were identified in the soybean OsDREB3 genome, which was integrated in chromosome 1 with an additional 394 bp insertion between soybean genomic DNA and exogenous gene. Based on these inserts and flanking sequences, the event-specific qualitative and quantitative PCR system was established for this line. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.1%. In the quantitative real-time PCR assay, the LOD and the limit of quantity were 10 and 100 haploid genome copies, respectively. The goodness of the linearity and high efficiency of the PCR reaction indicated the utility of the established PCR system. This study provides two reliable methods and information for detection, identification, and quantification of the presence of non-authorized transgenic soybean OsDREB3.     

Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products

An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10-10⁶ copies of each target.     

Establishment and evaluation of event-specific qualitative and quantitative PCR method for genetically modified soybean DP-356043-5

With the increasing development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. The polymerase chain reaction (PCR) technique has been the mainstay for GMO detection, especially for event-specific qualitative and quantitative PCR detection methods, which have become the internationally agreed state-of-art. This paper describes the character and event-specific quantitative detection method of DP-356043-5 (356043) soybean. In this research, the flanking regions were characterized by inverse PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right and left flanking sequences. In the qualitative PCR assay, PCR systems were established with the species-specific and event-specific primers, respectively. And event-specific primers were established on both right and left flanking sequences; the limit of detection (LOD) was both 0.05% (approximates to 42 haploid genome copies). In the quantitative TaqMan real-time PCR assay, we obtained standard curves with good linearity and relatively high efficiency of PCR. All the results indicated that the established event-specific qualitative and quantitative PCR systems for 356043 soybean in this study were reliable and suitable for 356043 soybean detection in mixed samples. Besides, based on the flanking sequence information we obtained, not only the qualitative and quantitative PCR system for detecting 356043 soybean can be established, but also some other novel event-specific detection methods using gene microarray, biosensor, etc., with target sequence on them can also be developed, which have a good value for detecting 356043 soybean.     

Development of an Event-Specific Detection Method for Genetically Modified Maize IE034 by Quantitative Real-Time PCR

The insect-resistant transgenic maize event IE034 has been proved to be one of the most commercially developed transgenic maize events in China. This study was aimed to develop a stable and reliable quantitative detection method to monitor this new transgenic maize event. Here, we developed a novel event-specific real-time PCR method for this genetically modified maize event IE034. The resulting 134 base pair (bp) amplicon was designed according to the 5′ junction of inserted sequence and flanking maize genome sequence. Standard curve of the IE034 5′ event-specific sequence showed good linear regression and high PCR efficiency when using the IE034 pure line samples as calibrator. The limit of detection (LOD) for the IE034 detection method was estimated at approximately 8 initial template copies, and the limit of quantification (LOQ) was estimated at about 40 copies. The accuracy of this quantitative real-time PCR method was verified by screening four mixed DNA samples with known levels of the IE034 event (5, 1, 0.5, and 0.23 %, respectively). The quantified biases deviated from 8.7 to ?12.2 %, and the relative standard deviation (RSD) ranged from 2.7 to 12.7 %. These data indicated that this new-developed IE034 event-specific real-time PCR method is suitable and reliable for the quantification of IE034 maize and its derivates.     

Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1?kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1?% for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event-specific real-time PCR detection systems were reliable, sensitive and accurate.     

Event-specific qualitative and quantitative PCR detection of genetically modified rapeseed Topas 19/2

The herbicide-tolerant transgenic rapeseed Topas 19/2 (synonym HCN92) has been approved for environmental release in Canada, Japan, Australia and the USA, and exported to a number of other countries as raw material. The purpose of this study was to establish event-specific qualitative and quantitative detection methods for Topas 19/2. The 3′-integration junction sequence spanning the host plant DNA and the integrated transgene of the Topas 19/2 event was isolated and identified. The event-specific qualitative detection method was established to produce an amplicon of 110 basepairs (bp) with an absolute detection limit of 10 initial template copies. The event-specific quantitative detection method was developed with the limit of detection (LOD) and limit of quantification (LOQ) being approximately 5 and 50 initial template copies, respectively. The developed real-time PCR systems were assessed using two mixed rapeseed samples with known Topas 19/2 contents. Expected results were obtained.     

Real-time PCR method for detection of the transgenic rice event TT51-1

The insect-resistant transgenic rice event TT51-1 (synonym BT63) has been found illicitly planted and distributed for years although it has never been approved for commercial cultivation in any country up to now. The purpose of this study was to establish a detection method that is specific for this transformation event. The event-specific PCR method produces an amplicon of 120 basepairs (bp) based on the revealed 3′ junction sequence with a limit of detection (LOD) and a limit of quantification (LOQ) being approximately 5 and 10 initial template copies, respectively. Two mixed rice samples with known TT51-1 contents were used to verify the developed real-time PCR system, from which the expected results were observed.     

The construction of pMD18-HT-Soybean as a calibrator plasmid and nested PCR assay for herbicide-tolerant soybeans

A multiple-target plasmid designated as pMD18-HT-Soybean, comprising part of a junction region of genetically modified soybean events A2704-12, A5547-127, MON89788 and GTS-40-3-2, and the endogenous soybean-specific lectin gene were constructed. The limit of detection for quantification of these four event-specific genes using pMD18-HT-Soybean plasmid was 20 copies. Furthermore, a nested PCR detection method was developed for the above four genetically modified soybean events. LOD value of nested PCR detection was 0.005 %. The above results demonstrated that the plasmid pMD18-HT-Soybean DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2, MON89788, A2704-12 and A5547-127 in food and feed products. And the validated results also indicated that the developed nested PCR method can be used for identification and quantification of four genetically modified soybean events and its derivates.