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.     

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     

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.     

Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize

The aim of this study was to trace selected nucleic acid and protein components of isogene versus Bt transgene maize within the bovine gastrointestinal tract (GIT). After feeding 22 cattle for 4 weeks with Bt176 maize, different plant genes and the recombinant protein CryIAb were quantified during digestion. Furthermore, a first initial characterization of rumen bacteria was approached, using 16rDNA gene sequencing comparing isogene- against transgene-fed animals. Ingesta samples of different GIT sections (rumen, abomasum, jejunum, colon) were analysed for chloroplast, maize invertase, zein and Bt toxin (CryIAb) gene fragments using quantitative real-time PCR. First, the initial gene dose of these maize genes was detected in maize silage. During digestion, a significant reduction of high-to-medium abundant plant gene fragments was shown depending on the dwell-time and the initial gene copy number. Immunoreactive CryIAb protein was quantified by ELISA in intestinal samples indicating a significant loss of that protein. Remarkable amounts of Bt toxin were found in all contents of the GIT and the protein was still present in faeces. For the first time, the influence of CryIAb transgene maize on rumen bacterial microflora was investigated compared to isogene material through analysis of 497 individual bacterial 16S rDNA sequences. In principle, specific bacterial leader-species could be identified in all bovine rumen extracts, but no significant influence of Bt176 maize feed was found on the composition of the microbial population. This investigation provides supplementing data to further evaluate the fate of novel recombinant material originating from transgene feed or food within the mammalian GIT.     

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.     

Quantitative,multiplex ligation-dependent probe amplification for the determination of eight genetically modified maize events

Specific 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 robust, quantitative, sensitive, nine-plex ligation-dependent probe amplification method, GMO-MLPA, for event-specific detection of maize TC1507, MON810, NK603, MON863, BT176, T25, GA21, construct-specific detection of BT11, and detection of the endogenous hmga maize reference gene. Ligated probes are amplified by PCR. Amplicons are detected using capillary electrophoresis. Specific GMO signals are normalised relative to the signal from the endogenous hmga gene and quantified by comparing with known standard curves. The method is suitable for quantification in the 0–2% range. Agreement was obtained in 149 of 160 determinations when 11 known mixtures of GMO and 9 food and feed samples were analysed using the GMO-MLPA method 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.