Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.     

Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.     

转基因大豆及其制品二重荧光定量PCR的建立与验证

研究选用花椰菜花叶病毒35S启动子(P-35S)和根癌农杆菌胭脂碱合成酶基因终止子(T-NOS)为靶标基因,运用多重实时荧光PCR技术对转基因大豆及其制品进行快速筛选检测。实验通过样品核酸提取与质控,多重引物及荧光探针的设计与筛选,反应条件和反应体系的对比优化,摸索出二重荧光定量PCR检测的最佳反应体系。同时通过特异性、重复性、灵敏性和适用性实验验证,确保了该方法在同时检测2个靶标基因时,无荧光信号的相互干扰,不会出现假阴性和假阳性结果。结果表明,方法特异性高,可同时筛查两个靶标基因,扩增效率在90%~110%之间,标准曲线决定系数R²>0.98,确定了最低检测限为2拷贝/μL。开发和建立的二重荧光定量PCR检测技术可以实现一管多检的实际需要,降低试剂成本,缩短检测时间,为大豆及其深加工产品转基因成分的快速检测提供了有效方法,为促进食品进出口提供技术保障。     

微生物酶制剂中转基因微生物的实时荧光PCR检测方法

针对微生物酶制剂中残留转基因微生物的检测问题,根据醇氧化酶-1(AOX1)启动子基因的序列信息设计一对引物及一条MGB探针,建立了转基因微生物的实时荧光PCR筛选检测方法.实验结果表明,该方法灵敏度达到1pg,可以准确判定生产线上不同分离阶段的微生物酶制剂中的转基因微生物残留情况.该方法准确度和灵敏度高,操作方便,可作为微生物酶制剂中转基因微生物分离状况的监测方法,也可为其他转基因微生物检测研究提供借鉴和参考.     

微生物酶制剂中转基因微生物的实时荧光PCR检测方法

针对微生物酶制剂中残留转基因微生物的检测问题,根据醇氧化酶-1（AOX1）启动子基因的序列信息设计一对引物及一条MGB探针,建立了转基因微生物的实时荧光PCR筛选检测方法。实验结果表明,该方法灵敏度达到1pg,可以准确判定生产线上不同分离阶段的微生物酶制剂中的转基因微生物残留情况。该方法准确度和灵敏度高,操作方便,可作为微生物酶制剂中转基因微生物分离状况的监测方法,也可为其他转基因微生物检测研究提供借鉴和参考。      

Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.     

转基因甜菜GTSB77品系特异性实时荧光聚合酶链式反应检测方法建立

目的 为实现转基因甜菜GTSB77的标识管理,建立其品系特异性实时荧光聚合酶链式反应(PCR)检测方法。方法 针对GTSB77的3′端外源插入片段与甜菜基因组DNA之间的邻接区序列设计引物和探针,建立GTSB77品系特异性实时荧光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GTSB77检测方法特异性强,定量限(LOQ)为16拷贝,扩增效率为102%,重复性测试结果相对标准偏差(RSD)介于0.21%～1.66%之间。结论 建立的实时荧光PCR方法可应用于GTSB77的鉴定检测。     

Development of a quadruplex-real-time-PCR for screening food for genetically modified organisms

With the ever increasing number of genetically modified plants authorized worldwide, including in the European Union, high throughput detection methods need to be developed. In this paper, a quadruplex-real-time-PCR method is described which allows rapid and simultaneous screening of food for the presence of target DNA sequences from the cauliflower mosaic virus 35S promoter, the NOS terminator from Agrobacterium tumefaciens, the soya reference lectin gene and the maize reference alcool dehydrogenase gene (adh). Three of the four primers and probe combinations have already been published elsewhere, whereas primers and probe for NOS terminator-PCR were developed in-house. Validation data show sensitivities down to five copies for 35S promoter and NOS terminator PCR, even when target sequences of the competing PCRs are in large excess. Thorough adjustment of primer and probe concentrations allowed high individual PCR efficiencies with negligible physical cross-talk between the four detection channels. This method provides a basis for a rapid screening of food for the most frequently used regulatory elements present in GM crops authorized for food in the European Union. In addition it provides information about the presence of species which are possibly genetically modified.     

食品中转基因大豆成分的定性和定量检测

为建立食品中转基因成分的定性定量分析，运用传统的定性PCR方法检测大豆加工产品中转基因成分的存在，运用Taqman探针技术，对存在的转基因成分进行准确定量。对于定量过程中由于扩增效率的不同造成的误差，通过大豆的内源基因Lectin进行校正，根据△Ct值对应于校正曲线计算出转基因大豆的含量。本方法灵敏度达到0．1％，误差范围小于30．0％。可用于转基因大豆的监测管理。     

Validation and collaborative study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products

In this work the intra and inter-laboratory validation of a duplex real-time PCR screening method for the detection of genetically modified (gm) plants is described. Target DNA sequences from Cauliflower Mosaic Virus 35S promoter (P35S) and nos-terminator from Agrobacterium tumefaciens (T-nos) are amplified. The duplex real-time PCR method is using primer and probe sequences that have already been published for the individual (“single”) detection of both target sequences. The validation showed sensitivity comparable to the single PCR standard methods. In addition, combined with a reference gene and using reference standard material, the method can be used to semiquantitatively estimate the amount of gm plants in an unknown sample.     

Analysis of caecal microbiota in rats fed with genetically modified rice by real-time quantitative PCR

The effect of genetically modified rice (GMR) on bacterial communities in caecal content was analyzed in a 90-d feeding rat model. A total of 12 groups of rats, which included male and female, were fed with the basal diets containing 30%, 50%, 70% GMR (B(1), B(2), B(3)) or 30%, 50%, 70% non-GMR (D(1), D(2), D(3)). The structure of intestinal microflora was estimated by real-time quantitative PCR (RQ-PCR) based on genus-specific 16s rDNA primers. SYBR Green was used for accurate detection and quantification of 6 kinds of major bacteria shared by humans and rats. According to RQ-PCR, the genome copies of Lactobacillus group from the cecum of male rats fed with 70% non-GMR was higher than those fed with 70% GMR and the relative abundance of Lactobacillus group also higher for group D. This result was in contrast with the E. coli subgroup, which was more numerous in proportion of group B, except D(2) and B(2) for male rats. The Clostridium perfringens subgroup was numerically more abundant in group D than group B of the same level, also except D(2) and B(2) for male rats. These results suggested that GMR had a complex effect on caecal microflora that may be related to the health of the host.     

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.     

Detection method for genetically modified papaya using duplex PCR

A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.     

实时荧光定量PCR技术在粮油转基因成分检测中的应用

随着转基因技术在谷物育种中的广泛应用,以及转基因粮油及其制品消费的不断增加,加强粮油转基因成分的检测技术的研究极为必要.阐述了实时荧光定量PCR技术在粮油转基因成分检测中的应用,主要从实时荧光定量PCR技术,实验室条件,实验步骤以及应用范围等方面进行了阐述.     

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.     

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.     

PCR技术在转基因食品检测中应用

该文在简述转基因食品安全性基础上,提出其检测方法—PCR技术,重点介绍PCR技术在转基因食品检测上应用及发展趋势。     

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X ₀) and the content of GM DNA in tested material (X _SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.     

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.