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.     

Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

Rapid real-time PCR detection of transgenic cry1C rice using plasmid molecule as calibrator

Transgenic Bacillus thuringiensis (Bt) rice expressing cry1C gene showed a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Till now, no detection method based on the plasmid molecule as the calibrator has been reported. In this study, one plasmid molecule containing the rice root-specific gene (gos9) endogenous sequence and the cry1C rice 5′ event-specific sequence was developed. Real-time polymerase chain reaction (PCR) method was established using the developed plasmid molecule as the calibrator. Two standard curves for gos9 and the cry1C rice 5′ event-specific sequence showed high PCR efficiency and good linear regression. Limit of quantification of the plasmid molecule in quantitative PCR assays was 40 copies. Biases for 5 and 0.25 % content samples’ quantification were ?6.01 and ?3.55 % with acceptable standard deviation and repeatability standard deviation, respectively. Comparing with genomic DNA, the plasmid molecule was suitable for cry1C rice quantification as the calibrator. Furthermore, the present study provided a reliable and stable identification and quantification system for monitoring cry1C rice.     

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.     

Event-specific qualitative and quantitative detection of transgenic rice Kefeng-8 by characterization of the transgene flanking sequence

The transgenic rice line Kefeng-8 harboring insect-resistant genes Cry1Ac and CpTI showed ideal field performances characterized by high resistance to rice stem borer (Scirpophaga incertulas) and leaf roller (Cnaphalocrocis medinalis). This GM rice line is likely to be approved in China in the near future. In this study, we estimated the insert number of foreign genes, analyzed the flanking sequences of T-DNA in rice genome, and presented an event-specific detection method for this line. The results show that the foreign gene inserted one copy between position 11,774 and 11,805 of chromosome 11 (AC120536.5) in Kefeng-8 genome. Based on these inserts and border sequences, the event-specific qualitative and quantitative PCR system was established for Kefeng-8. The qualitative detection limit was assessed to be 0.1%, and the limit of quantitative method was assessed to be 100 initial template copies. Two mixed rice sample with known Kengfeng-8 contents were used to verify the quantitative method, from which the expected results were observed. This study provides a reliable method and information for detection, identification, and quantification of the presence of GM rice Kefeng-8.     

基于微滴式和芯片式数字PCR技术的转基因克螟稻成分的定量检测

以转基因克螟稻品系为研究对象,通过大米内源蔗糖磷酸合成酶基因和转基因克螟稻品系特异性序列的绝对拷贝数分析,建立转基因克螟稻成分的双重数字聚合酶链式反应（polymerase chain reaction,PCR）定量分析方法。本研究中转基因克螟稻定量体系中最适DNA添加量在10 pg～13 ng之间,模板断裂程度、PCR扩增退火温度等因素对定量结果的影响不大。其定量的绝对灵敏度达1 copies/μL,可在微滴式和芯片式数字PCR平台上准确检测质量分数在0.1%～100%之间的转基因克螟稻成分,尤其是对低于1%的样品,其定量准确性高于传统的实时荧光PCR方法。本研究建立的转基因克螟稻成分定量分析方法适用性较好,可用于转基因水稻规范化与标准化的定量分析。     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng?6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng?6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng?6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95?%) between 0.07 and 0.52, a PCR efficiency of 105?% and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng?6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng?6 DNA were observed.     

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.     

常见转基因大米数字聚合酶链式反应多重定量检测方法研究

目的 建立4种转基因(genetically modified, GM)大米成分多重定量检测方法, 解决混合转基因产品和多品系杂交的多价转基因产品的高通量精准定量难题。方法 采用Raindrop微滴式数字聚合酶链式反应(polymerase chain reaction, PCR)方法对4种常见转基因大米TT51-1、克螟稻、科丰6号、M12品系特异性基因和大米蔗糖磷酸合成酶基因逐一进行拷贝数分析, 并将其转化为含量百分比。结果 转基因含量在0.1%~100.0%范围内的4品系转基因大米混合样品的定量体系线性关系良好, 标准曲线r2值均在0.99以上, 定量相对灵敏度可达0.1%, 相对误差和相对标准偏差值均小于25%, 定量准确性和精密度符合国际通用要求。结论 本研究成功建立了4种常见转基因大米成分的多重定量检测方法, 解决了常规方法一次只能定量分析单一样品的缺点, 为大米、稻谷及其初加工产品中多种转基因成分的高效定量提供了新的技术手段。     

Event-specific detection system of stacked genetically modified maize by using the multiplex-PCR technique

Stacked genetically modified (GM) crops are becoming popular for their enhanced production efficiency and improved functional properties. In this study, we developed an event-specific PCR method for simple qualitative detection of stacked events combining more than 2 transgenic traits. Ten primer sets were designed, including 9 that were event-specific and 1 that was specific for a maize endogenous gene. Five event-specific multiplex-PCR systems were built, based on the main type of stacked GM events approved in Korea. Multiplex PCR was performed with mixtures of template DNA extracted from certified reference materials. PCR amplicons (3 or 4 by type) of expected sizes and mutually similar intensities were detected. The limit of detection was approximately 0.1%(v/v) for stacked GM maize in all event-specific PCRs. This method may be useful for the specific detection and monitoring of stacked GM maize lines and individual parent GM maize lines, by effectively distinguishing genestacked events.     

土壤磷素高效利用转基因大豆特异性PCR检测方法

华南农业大学根系生物学研究中心采用拟南芥的紫色酸性磷酸酶基因AtPAP15转化大豆品系粤春03-3（YC03-3）,获得了酸性磷酸酶活性明显提高、可高效利用土壤磷素的转基因大豆新品系AP15-1。本研究以AP15-1为研究对象,应用TAIL-PCR技术,根据载体序列设计特异引物,获得了转化载体左侧插入的旁邻序列。设计事件特异性检测引物,进行PCR扩增,只能在AP15-1的样品中扩增出特异性条带,进一步用实时荧光定量PCR作分析,结果显示,该引物对重复性好,融解曲线显示只有一个特异峰值。本实验应用该引物对建立的检测方法,检测的灵敏度可以达到0.01%,实时荧光定量PCR检测的极限值可以达到9个基因组的拷贝数,能够满足对转基因大豆新品系AP15-1及其衍生品种检测的需要。     

应用多重PCR-液相悬浮芯片技术检测转基因水稻品系

开展转基因水稻的多重聚合酶链式反应（polymerase chain reaction,PCR）液相芯片检测方法研究。针对科丰6号（KF6）、科丰8号（KF8）、华恢1号（BT63）、克螟稻（KMD1）、M12、T1C-19、T2A-1、LL62和LL601九种转基因水稻的侧翼序列,设计合成了在生物素标记的多重PCR扩增引物与固定在荧光编码微球上的探针,建立了2 个多重PCR-液相芯片检测体系,可同时检测出这9 种转基因水稻成分。结果表明,9 种转基因水稻的引物和探针都具有较高的特异性,各组引物/探针之间无交叉扩增和非特异杂交,且在多重PCR-液相芯片检测中9 种转基因水稻品系的相对检测灵敏度达到0.1%水平,符合欧盟和其他国家有关转基因产品标识的要求。本方法的检测效率和准确性均高于传统方法,可作为进出口转基因产品和国内转基因检测的有效方法。     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng 6 DNA were observed.     

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.     

Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.     

Detection of genetically modified rice: collaborative validation study of a construct-specific real-time PCR method for detection of transgenic Bt rice

A collaborative trial study has been conducted for validation of an extraction method and a subsequent real-time PCR for detection of a transgenic Bt rice line (‘Bt63’) in rice products originating from China. A total of 17 laboratories participated in the study and each laboratory received 16 coded samples comprising of rice grain flours, rice noodle flours and plasmid DNAs. Of the accepted results all Bt63-positive rice grain samples (0.1 or 0.05% w/w) and all rice noodle samples prepared from marketed rice products were detected correctly. The result demonstrates that ‘Bt63’ rice is detectable even at low relative mass concentrations of 0.05%. The absolute LOD determined with plasmid DNA samples showed to be at least five copies of the ‘Bt63’ target sequence. The data provided in this study show that the method is fit-for-purpose to inspect Chinese rice products for the presence of EU-unauthorised rice lines carrying the ‘Bt63’ construct.     

Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6 by characterization of the transgene flanking sequence

Using TAIL-PCR (thermal asymmetric interlaced PCR), we analyzed the flanking sequences of transfer DNA (T-DNA) in transgenic rice Kefeng-6, which has insect-resistant genes, Cry1Ac and CpTi. Two junction sequences of T-DNA were identified in the Kefeng-6 genome: one integrated in chromosome 6 with an additional 22-bp insertion and another one in chromosome 9 with a 4-bp insertion between rice genomic DNA and T-DNA. Based on these inserts and border sequences, the event-specific qualitative and quantitative PCR system was established for this line. The relative limit of qualitative detection was assessed to be between 0.1 and 0.05%, and the absolute limit of detection in the quantitative PCR was approximately five initial copies. Two mixed rice samples with known Kefeng-6 contents were used to verify the quantitative method, from which the expected results were observed. This study provides a reliable method and information for detection, identification, and quantification of the presence of non-authorized GM rice Kefeng-6.     

Development of a real‐time multiplex PCR system for the quantitative detection of Chinese GM rice

BACKGROUND: Rice is one of the most important staple food crops for more than half the global population, who rely on it for as much as 80% of their diet. By one estimate, the world population is projected to grow to approximately 11 billion people by the year 2050. So it is a formidable task to meet the future demand. For this reason, breeders make a great deal of effort to produce new rice varieties with traits such as higher yield, improved nutritional content and better resistance to disease and pests, via transgenic biotechnological protocols. Dozens of transgenic rice lines have been developed since the first transgenic rice plant production in the late 1980s. With the rapid approach of transgenic rice commercialisation, it is becoming necessary to develop techniques capable of detecting and quantifying genetically modified (GM) rice. RESULT: Here we describe a method in which transgenic DNA is quantified by amplifying part of the 35S‐CaMV promoter and standardising it against an amplified portion of an endogenous single copy, rice specific gene encoding sucrose phosphate synthase. Both reactions are performed simultaneously in a single tube. Standard calibration curves were developed by diluting DNA extracted from a blend of non‐transgenic (c.v., Nipponbare) and 5% KMD2 transgenic rice. The method was tested for the quantification of the five GM rice events, including KMD2, Wan 21A, GC‐1, H1597 and TR4, which contain the 35S‐CaMV promoter. The coefficient of variation varied from 3.15% to 12.84%, which is up to acceptance criterion over the dynamic range of the method. CONCLUSION: In this study, we successfully applied a multiplex real‐time PCR assay to GM rice, which employed SPS as the endogenous reference gene and the gene regulation element 35S‐CaMV promoter as a GMO marker. The detection limit and limit of quantification is sufficient to comply with all relevant regulations in the EU and worldwide. The detection system could be applied in routine analysis for the quantification of GM rice in food materials, such as instant rice, unpolished rice, rice flours, biscuit powders, and starch. It may prove useful with regard to a robust screening technique of broad utility as transgenic rice enters global commodity markets. Copyright © 2009 Society of Chemical Industry     

转基因水稻Bt63的外源基因相对含量分析

为分析转基因Bt63稻米外源基因含量,利用新型、灵敏和高通量的实时荧光定量PCR技术,以RBE-4作为转基因水稻的内源参照基因,通过梯度稀释法,分别获得了Bt 63和RBE-4基因的Ct值与起始模板相关性的标准曲线,相关系数分别为0.996 6和0.995 4。通过转基因水稻外源基因(Bt63)和内标基因(RBE-4)起始模板数的比较,测定了外源基因在转基因水稻中的相对含量,这项研究将为建立转基因水稻抗虫能力评价体系和转基因稻谷的流通和监管提供技术支撑。     

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.