Monitoring of Bt11 and Bt176 genetically modified maize in food sold commercially in Brazil from 2005 to 2007

BACKGROUND: The first genetically modified (GM) maize lines were approved for trading in Brazil after December 2007 and they were T25, MON810, Bt11, NK603 and GA21. The polymerase chain reaction (PCR) method was employed to monitor the presence of Bt11 and nested PCR was used to detect the presence of Bt176 in 81 maize‐derived products (maize flour, corn meal, maize flour flakes and polenta) that were sold in Brazilian market from 2005 to 2007, before the release of GM maize in Brazil. RESULTS: The PCR detection limit for Bt11 was 10 g kg^?1 and for nested PCR of Bt176 it was 1 g kg^?1. All Brazilian samples analyzed showed no positive signal for these GM maize events. CONCLUSION: Bt11 and Bt176 GM maize lines were not detected by specific PCR in 81 maize‐derived food samples sold in Brazil from 2005 to 2007, before the commercial release of GM maize in Brazil. These Brazilian food industries were in compliance with the rules stipulated by the current legislation with respect to consumer requirements about GMO labeling. Copyright © 2010 Society of Chemical Industry     

The effect of ensiling on PCR-based detection of genetically modified Bt maize

 The detection of the genetic modification in silage obtained from insect-resistant Bt maize by means of the polymerase chain reaction (PCR) is described. The detectability of the transgene was shown to be dependent on the length of the genomic target sequence chosen for amplication by the PCR. By amplifying a Bt-maize-specific DNA sequence of 211 bp the genetic modification was detected up to 7 months after ensilage. The effect of maize DNA degradation in the course of the ensilage on the detectability of target sequences was demonstrated in model experiments. Received: 17 December 1998     

PCR-based quantification of genetically modified Bt maize: single-competitive versus dual-competitive approach

 Methods for the quantification of transgenic insect-resistant Bt maize by single- and dual-competitive PCR were developed. The analysis of mixtures of DNA solutions, as well as of maize flours containing defined amounts of Bt maize, demonstrated the usefulness of single-competitive PCR based on coamplification of the CDPK promoter/cryIA(b) gene region of Bt maize and an internal standard. Upon heat treatment of DNA solutions and maize flour, respectively, the recovery of the Bt proportion compared to the starting material determined by single-competitive PCR decreased significantly. This systematic error could be compensated for by using a dual-competitive approach based on PCR quantification of the transgenic target sequence of Bt maize and of the maize specific invertase gene (ivr1). Received: 24 January 2000 / Revised version: 25 February 2000     

Detection of genetically modified insect-resistant Bt maize by means of polymerase chain reaction

 A method for the detection of genetically modified insect-resistant maize expressing a synthetic gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis is described. The procedure includes: (1) extraction of genomic DNA, (2) amplification of the inserted synthetic gene by means of the polymerase chain reaction (PCR) method, and (3) characterization and confirmation of the PCR product by Southern hybridization with a digoxigenin-labelled oligonucleotide probe and DNA sequencing. Received: 18 February 1997 / Revised version: 27 March 1997     

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.     

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.     

Screening of genetically modified organisms and specific detection of Bt176 maize in flours and starches by PCR-enzyme linked immunosorbent assay

Polymerase chain reaction (PCR)-enzyme linked immunosorbent assays (ELISAs) targeting either the 35S promoter or the Bt176 specific junction sequence were developed to screen for the presence of genetically modified organisms (GMOs) and specifically detect Bt176 maize in flours and starches. Two additional PCR-ELISA assays were developed to validate the results: one, based on the detection of the maize alcohol dehydrogenase 1 promoter specifically detected the presence of maize, and the other, based on the detection of a conserved sequence of plants ( 26S ribosomal RNA gene), validated the extracted DNA amplification. The PCR-ELISA assays developed here were highly specific and found to be as sensitive as the reference Southern hybridisation assay. The PCR-ELISA tests were at least 6 times more sensitive than gel electrophoresis and allowed 0.1% GMOs to be detected in Bt176, Bt11, Mon810 maize and Roundup Ready soybean. The PCR-ELISA tests are a method of choice for GMO screening and identifying Bt176 maize in flours and native starches. They may offer a cheaper alternative to the expensive real-time PCR assays and may be useful in laboratory GMO monitoring.     

Detection of recombinant DNA from genetically modified papaya

A method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) papaya (55-1 line), of which the mandatory safety assessment has not been finished in Japan because of insufficient data. The papaya intrinsic papain gene was used as an internal control. The results of PCR amplification of the papain gene segment indicated that a commercial silica membrane type kit (QIAGEN DNeasy plant mini) was useful for extraction of DNA from papaya fruit, but not for extraction from canned papaya fruit. On the other hand, a commercial ion-exchange type kit (QIAGEN Genomic-tip) provided enough purified DNA for PCR from canned papaya fruit. Compared with the parental line and other commercial non-GM papayas, the DNA from GM papaya fruit provided specific amplification bands in PCR with five primer pairs (Nos. 2-6) including beta-glucuronidase and neomycin phosphotransferase II gene-specific ones. On the other hand, the primer pairs recognizing these genes showed false-positive results when we used DNAs from canned papaya. Therefore, we recommend that the primer pairs (Nos. 5 and 6) recognizing the sequences derived from two different species of organism should be used in order to detect specifically the GM papaya in canned fruits.     

转基因玉米 BT11 品系环介导等温扩增(LAMP)检测方法的建立

目的 建立转基因玉米 BT11 品系的环介导等温扩增(LAMP)检测方法。方法 根据转基因玉米 BT11 品系特有序列(AY123624.1 和 AY629236)设计引物, 优化建立 LAMP 反应体系, 对该体系进行特异性、 灵敏度、 稳定性分析。结果 本研究设计的引物具有很好的特异性, 可特异性检测出转基因玉米 BT11 品系; 检测灵敏 度可达 0.5%; 经精密度实验分析, 该方法的稳定性良好。结论 LAMP 方法检测转基因玉米品系 BT11 具有特 异性高、稳定性好、快速灵敏、过程可视等优点, 具有广阔的应用前景。     

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).     

Detection of genetically modified soya and maize: Impact of heat processing

The analysis of processed foods entails a number of complications, which negatively affect the performance of DNA based detection methods. Heat-processing methods viz. autoclaving and micro-waving, that mimic processing and manufacturing, as model unit operation systems were used to study their effect on the detection of genetically modified organisms (GMOs). This study confirms the premise that high temperature and/or pressure significantly reduce the level of detectable DNA. PCR methods were developed and adapted to target varying amplicon sizes of the trait, construct and event specific gene sequences that occur in MON-810 maize and Roundup Ready^® soybean. Integrity of DNA, recovery and PCR amplicon size (<200 bp) are major factors that direct the successful detection of GMOs in processed foods. The model systems used provide a platform to devise better strategies in developing detection protocols, especially for processed foods containing GMOs.     

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     

转基因玉米成分定性检测能力验证结果与分析

目的提高检测实验室对转基因玉米成分的检测能力和检测人员专业技术水平,增强实验室竞争力。方法中心实验室依据能力验证作业指导书和农业部公告的要求,参与了ACAS-PT570(2018)粮食转基因检测能力验证测试,分别对编号18-F750和18-K388样品进行PCR检测。结果 18-F750和18-K388待测样品中, CaMV35S启动子、FMV35S启动子、NOS终止子、Bt基因、CP4 EPSPS基因、bar/pat基因、59122转化体、MIR162转化体和Bt11转化体均为阳性。结论本次能力验证获得满意评价。     

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.     

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.