Laboratory-performance study of quantitative PCR methods to analyze an approved genetically modified maize (Mon810 Line)

A laboratory-performance study was carried out to investigate factors affecting the reliability of the quantitative PCR method to analyze an approved genetically modified (GM) maize (Mon810 line). Test maize powdered samples were prepared as blind samples containing a high (assigned value; 5.45%) or low (assigned value; 0.35%) concentration of the Mon810 line. After confirmation of their homogeneity, they were provided to 27 laboratories participating in the collaborative study. The data were collected from all laboratories and statistically analyzed. Two laboratories, which used a Roche LightCycler (LC), reported significantly high test values. A further examination showed that the LC method is greatly affected by the equipment itself or PCR reagents, resulting in poor repeatability. On the other hand, some laboratories, which used ABI quantitative PCR equipment, reported erroneous test values. In these laboratories, the errors appeared to have been due to inadequate quality and/or yield of DNA. To identify factors affecting the test values, analysis of the measured values for the taxon-specific gene will be useful. Furthermore, the modified silica-gel membrane DNA extraction method made it possible to extract the required amounts of DNA more easily and in a shorter time than before.     

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.     

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 and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.     

Quantitative competitive PCR for the detection of genetically modified soybean and maize

 The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques. Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more than 1% RRS were determined by QC-PCR using various amounts of standard DNA. Received: 13 January 1998 / Revised version: 4 March 1998     

Qualitative and event-specific PCR real-time detection methods for StarLink maize

In this paper we present qualitative detection and identification methods for StarLink maize (event CBH-351). The methodology proposed envisages detection of an internal target site in the cry9c coding region, as well as two event-specific target sites at the junction between the CBH-351 insert DNA and the genomic plant DNA. The cry9c-specific primer pair, generating a 180 bp amplicon, has been tested and optimised for conventional end-point PCR amplification. The event-specific primer pairs, generating amplicons of 138 bp and 100 bp respectively, give good performance in a conventional end-point PCR and in a real-time PCR assay. Our results clearly demonstrate that the primer pairs proposed can be used in an unambiguous and specific PCR identification assay for StarLink maize.     

Simple,sensitive, accurate multiplex quantitative competitive PCR with capillary electrophoresis detection for the determination of genetically modified maize

Legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a simple and accurate capillary electrophoresis multiplex quantitative competitive PCR (ce-mqcPCR) method for event-specific quantification of the five novel GM maize events DAS59122, LY038, MON88017, MIR604 and Event 3272. The method combines the simplicity of constructing multiple competitors in silico with the high resolution and sensitivity of fluorescence capillary electrophoresis and the use of an internal template reference amplicon. The competitors are synthesised commercially and added in equal amounts as a restriction enzyme-digested plasmid insert to the multiplex PCR. Quantification is performed by analysing the relative amounts of GMO and GMO competitor fragment pairs after capillary electrophoresis and correcting for differences in maize DNA by comparing with the internal reference gene amplicon. Since the competitors employ the same primers as their corresponding targets, all existing qualitative multiplex PCRs can in principle easily be converted to quantitative assays without changing primer sets or amplification conditions. The ce-mqcPCR method correctly determined 120 GMO templates in known mixed samples. No false-positive or false-negative signals were obtained.     

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.     

转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.     

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.     

Detection system of stacked genetically modified maize using multiplex PCR

A multiplex polymerase chain reaction (PCR) method was developed to identify and distinguish 3 kinds of stacked genetically modified (GM) maize (MON810× MON863, NK603×MON863, and NK603×MON810× MON863). Four primer pairs, SSIIb JHF/JHR, C3b 5′/TAP1–3′, HS01/cry-CR01, and HS01/CTP164-3′ yielded 101, 129, 194, and 314 bp amplicons, respectively, Using the genomic DNA of the 3 stacked GM maize as templates, 3 or 4 corresponding PCR amplicons were amplified with similar band intensities by the multiplex PCR. The limit of detection (LOD) was approximately 0.5% for 3 kinds of stacked GM maize, using the multiplex PCR. The detection system using multiplex PCR developed in this study may be applicable to monitoring, identifying, and distinguishing not only the stacked GM maizes but also other stacked genetically modified organisms (GMOs).     

Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNA from two genetically modified organisms

The application of a ligation-dependent probe amplification (LPA) technique to the simultaneous event-specific detection and relative quantification of DNA from genetically modified organisms in foods is described. The system is based on the ligation of synthetic bipartite probes when hybridized to the corresponding target DNA sequence. The ligation products possess lengths characteristic for each target sequence. Universal primer binding sites (PBS) at the 5′ and 3′-ends enable their subsequent competitive amplification using one common pair of primers. The use of one fluorescein (FAM) labeled primer permits amplification products to be separated and detected via capillary electrophoresis. Respective probes were designed to allow the detection of reference genes in the genomes from maize and soya, as well as of event-specific junction regions in the transgenic maize line MON810 and in Roundup Ready soya. Specificity, sensitivity, and the potential of the technique for the relative quantification of recombinant DNA were assayed using mixtures of DNA extracted from certified reference maize and soybean flours. The novel strategy results in a modular system which can be complemented by further probes to broaden the range of target sequences.     

Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0–2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5′-sequence and a GMO specific 3′-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is therefore suitable for quantification purposes for food and feed containing the most common maize GMOs.     

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.     

快速定量检测转基因番茄方法的研究

本文以转基因番茄"华番一号"为原材料,采用了特异性的引物和探针,对构建的质粒载体进行转基因番茄的实时荧光PCR定量检测.其相对检测灵敏度可达到6个拷贝.可用于转基因番茄的定性和定量检测.     

A detection method for recombinant DNA from genetically modified maize CBH351

A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize CBH351, which has not authorized as safe for use in foods and feeds in Japan yet. We analyzed a recombinant DNA (r-DNA) sequence introduced into CBH351 maize and designed specific primer pairs to amplify a segment including part of the r-DNA. The PCR products obtained by using the designed primer pairs are specific for CBH351 and should prevent false positive results caused by other maizes and other main cereal crops. The r-DNA introduced into CBH351 could be detected from maize samples containing 0.05-0.1% CBH351 maize. This sensitivity is theoretically equivalent to a level of several genome copies and so this technique is a very efficient means to detect CBH351 maize.