Detection of eight genetically modified canola events using two event-specific pentaplex PCR systems

An event-specific multiplex polymerase chain reaction (PCR) detection method was developed to simultaneously detect eight genetically modified (GM) canola events (GT73, MS1, MS8, RF1, RF2, RF3, T45, and Topas 19/2). For a successful multiplex PCR assay, the eight GM canola events were divided into groups 1 and 2 in consideration of their amplicon sizes, primer efficiencies, and target sequences. In addition to the canola endogenous reference gene, FatA, the two pentaplex PCR assays targeted group 1, containing GT73, MS8, RF3, and T45, and group 2, including MS1, RF1, RF2, and Topas 19/2. Event-specific primers targeting the eight GM canola events were designed, and their specificities were confirmed using 14 GM events of maize, soybean, cotton, and canola. After optimizing the reaction conditions, the limits of detection of these two assays were approximately 0.025% for group 1 and 0.0125% for group 2. This multiplex PCR method for eight GM canola events was validated by two operators, and the data confirmed the reliability of the developed assays. The method was used to test commercially available canola seed (eight samples) and meal (one sample) produced in South Korea, China, Canada, and Australia, and the results revealed one or more GM canola events in seven of the nine samples tested. These results show that the developed multiplex system is applicable for use in the specific testing of eight commercially available GM canola events.     

Crop-specific GMO matrix-multiplex PCR: A cost-efficient screening strategy for genetically modified maize and cotton events approved globally

Crop-specific GMO matrices of 199 genetically modified (GM) events, comprising 143 GM maize events with 75 genetic elements and 56 cotton events with 45 genetic elements, were developed to screen globally approved GM maize and cotton events. As per the compiled information in the matrix, frequently present genetic elements were identified using GMOSeek algorithm: eight genetic elements ([P-35S] [r-act] [T-35S] [T-nos] [pinII] [pat] [aad-1] [cp4 epsps]) in maize and four ([P-35S] [T-nos] [pat] [nptII]) in cotton. Based on the cost-efficiency, feasibility of plexing and coverage of GM events, maize-specific tetraplex PCR, targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), Cauliflower Mosaic Virus 35S terminator (T-35S) and endogenous alcohol dehydrogenase (Adh1) gene, with limit of detection (LOD) up to 0.5% was developed. For screening of GM cotton events, pentaplex PCR, targeting P-35S, T-nos, neomycin phosphotransferase II (nptII), phosphinothricin acetyltransferase (pat) and endogenous stearoyl-ACP desaturase (Sad1) gene, with LOD up to 0.25% was developed. Practical applicability of multiplex PCR assays was confirmed with six maize samples of proficiency testing and eight spiked cotton samples. The reported tetraplex and pentaplex PCR assays could efficiently screen 94% of maize and 93% of cotton events approved globally. The developed GMO matrices in combination with multiplex PCR could facilitate checking the GM status of seed samples or food and feed products, and monitoring for presence of authorized GMOs in food and supply chain. This approach can be easily employed by low resource GM testing laboratories in the developing countries, as the multiplex PCR assays are easy to operate with less time and cost inputs. The GMO matrices being presented herein are based on the current information of approved GM events of maize and cotton, which can be further upgraded by including new approved GM events. As most of the newly approved GM events are stacked versions of already commercialized GM events, the developed multiplex PCR assays could also be employed to screen for their presence.     

Monitoring of genetically modified soybean events in sausage products in South Korea

The use of genetically modified (GM) ingredients in various types of food products has increased worldwide. In this study, qualitative real-time PCR assays targeting five GM soybean events (RRS, MON89788, A2704-12, A5547-127, and MON87701) were performed to monitor their use in sausage samples. Thirty sausage samples containing soybean ingredients on their label were collected from Korean food markets and analyzed. The endogenous lectin gene of soybean was used as an internal positive control. Real-time PCR showed that the threshold cycle (Ct) values ranged from 28.62 to 33.90 for the 30 sausage samples. Of the 30 sausage samples, 19 samples were negative for the presence of GM soybeans. In the positive samples, the results revealed three GM soybean events [Roundup Ready Soybean (RRS), A2704-12, and/or MON89788] in 11 of the 30 food samples tested. These results demonstrate that sausage products contain different GM soybean events.     

Detection of unapproved genetically modified potatoes in Korea using multiplex polymerase chain reaction

Several genetically modified (GM) potato cultivars with improved traits such as increased amylopectin levels, decreased asparagine levels, and reduced incidence of black spot have been developed. In this study, we describe a multiplex polymerase chain reaction (PCR) method specific for four GM potato events (EH92-527-1, AM04-1020, PH05-026-0048, and E12) that are unauthorized in Korea. The UDP-glucose pyrophosphorylase (UGPase) gene was used as the endogenous reference gene. The specificity of the primer sets was evaluated using GM potatoes and other GM crops. The limit of detection in the developed multiplex PCR was confirmed to be approximately 0.04% (w/w). Thirty-three commercial products containing potato ingredients were tested by the multiplex PCR assay, and GM potato event E12 was found in one of the 33 samples. This result suggested that the developed PCR method could be used to effectively identify unauthorized GM potatoes in Korea.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.     

GMO matrix: A cost-effective approach for screening unauthorized genetically modified events in India

Use of a pragmatic, affordable and reliable approach for screening and detection of a large number of genetically modified (GM) crops/events is the need of hour. A cost-effective matrix approach to check the GM status of food/feed products and for screening the presence of authorized and unauthorized GM events in India is being reported in the present study. A genetically modified organism (GMO) screening matrix, with the information on 106 genetic element targets for detection of 141 GM events of 21 crops, is being presented. These include commercially cultivated Bt cotton events and other GM events, under field trials during the past six years (2006–2012) in the country. The information on GM events, which were either indigenously developed or imported for research purposes, is also presented in brief. Ten most frequently present targets, viz., [P-35S] [T-nos] [Os-Msca1] [cry1Ab] [cry1Ac] [cry1C] [cry2Ab] [GA20 oxidase1] [nptII] [bar], were identified to screen these events using a GMOseek algorithm. This user-friendly screening tool is flexible for further updates with the new GM events and targets/elements. The data reported here related to the GM crops/events in India and the related GMO matrix are valuable tools to assist in the detection of accidental presence of unauthorized GM events in the food and supply chain globally, as well as in the context of the new labelling requirements for food commodities, as per the amendment to enforce GM food labelling from January 2013 in India. The reported GMO matrix approach would facilitate efficient, rapid and cost-effective preliminary screening by eliminating the need for development of specific testing methodologies for each GM event.     

Traceability of genetically modified Roundup Ready soybean: A case study on sampling and analytical uncertainty along processing chain

A study on the fate of Genetically Modified (GM) Roundup Ready soybean (RRS) was undertaken on the following products: flour, protein flour, lecithin, crude and refined oil, broken grain, hull and expander of an industrial soybean manufacturing plant, with the aim to evaluate the possible effects of processing on the reliability of control plans. A sampling control plan was applied to all the products of the industrial manufactory plant. The best sampling point was identified based on the lowest impact of the analytical and sampling uncertainty.The best “fit for purpose” sampling point for the accurate evaluation of the Genetically Modified Organism (GMO) concentration measurement was identified in the processed products, e.g. flour and protein flour, thanks to the homogeneity on RRS in the batch and the better yield and quality of the extracted DNA.This study presents a practical approach to assess the two main factors that affect the reliability of the control plans: analytical and sampling uncertainty. The work was undertaken on GM soybean derived products, nevertheless the conclusions we reached could be also applied to verify compliance with GMO labelling threshold.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.     

Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event

Genetically modified (GM) canola is the most widely grown oilseed crop in Canada. At this time, commercially produced GM canola cultivars in Canada have the events GT73/RT73 and Ms8xRf3. Commercial seed sale of canola cultivars containing the GM events such as OXY235 and T45 has been discontinued. Adventitious presence of GM seeds and grains in non-GM grains is a concern for international grain trade, and development of effective detection methods is important. A multiplex qualitative PCR procedure was established for the detection of the GM canola events OXY235, Ms8xRf3, T45 and GT73. The presence of the GM canola events was also successfully detected in ground spiked wheat and barley grain samples prepared at 0.1%, 0.5% and 1.0% levels (w/w). The GT73 real-time PCR assay was successfully used to quantify DNA extracted from spiked ground canola samples consisting of 5%, 1%, 0.5% and 0.1% GT73 (w/w).     

Transboundary movement of genetically modified organisms in India: Current scenario and a decision support system

Genetically modified (GM) crops have benefited global agriculture by introduction of superior traits for better agronomic performance, ensuring nutritional security and mitigating climate change. In India, to meet the demand of burgeoning population and to withstand the changing climate, GM crops would play an important role. Since 1997, GM crops are being imported through Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, the designated nodal organization for quarantine processing and import of GMOs (referred to GM planting material in present context) for research purposes. In the present study, an attempt has been made to analyze the trend of import of GMOs. Till the end of 2015, 205 consignments of fifteen GM crops have been imported from 19 countries by public and private sector. Detailed analysis of diversity in traits of imported GM events and imported stacked traits in cotton and maize has been made. In the recent past, four consignments of GMOs have been exported for research purposes. Involvement of public/private sector in transboundary movement of GMOs was evaluated. Along with quarantine processing of imported/exported GMOs, molecular testing for specific transgenic elements as claimed by the importer/exporter is also carried out employing polymerase chain reaction (PCR) and real-time PCR based markers. Efficient detection strategies based on GMO matrix as a decision support system, loop-mediated isothermal amplification and multi-target real-time PCR-based systems have been developed. The data presented herein would provide a decision support system to check for authorized/unauthorized GMOs in food and supply chain.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

DNA检测技术及其在微生物采油中的应用

专题论文。第一节简述了聚合酶链式反应(PCR)技术的特点、原理及在微生物采油中的作用。第二节介绍了菌种16S rRNA基因碱基序列分析的操作程序，包括基因扩增及精翻、16S rRNA基因克隆、荧光标识基因序列PCR扩增、基因碱基序列及微生物属种确定，给出了2个MEOR菌的基因碱基序列及属种。第三节介绍了多酶切和混合酶切两种PCR．RFLP基因检测技术(RFLP：限制性片段长度多态性)，后一种方法使操作减少l／4。第四节介绍了直接DNA扩增基因检测技术，包括平板菌落法(检测时间2d)和最大可能数法(当天得检测结果)，DNA模板制备及其反应体系，电泳法检测及菌种检出。第五节介绍了DNA基因检测技术在吉林油田多项微生物采油矿场试验中的应用，包括常在菌对微生物采油影响分析．微生物单井吞吐、微生物清防蜡、微生物调驱效果分析，多功能案采油菌构建的实验探索。图2参9。     

Rapid multiresidue and multi-class screening for antibiotics and benzimidazoles in feed by ultra high performance liquid chromatography coupled to tandem mass spectrometry

An analytical strategy was developed for high-throughput screening of multiple antibiotics and two benzimidazoles in feed. Generic sample processing was applied without any purification step. After methanol extraction, the samples were centrifuged, concentrated, and analysed by ultra-high-performance liquid chromatography hyphenated to tandem mass spectrometry in the multiple reaction monitoring mode. Qualitative validation was carried out for more than 50 antibacterials of various classes, including aminocoumarin, amphenicols, beta-lactams, lincosamide, macrolides, diaminopyrimidine, quinolones, sulfonamides, streptogramin, pleuromutilin, polypeptide, quinoxaline, and tetracyclines, and also some benzimidazoles in feed at μg/kg level. Validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/CE for qualitative screening methods.This convenient, reliable, and sensitive method has been used successfully to monitor antibiotic residues in feeds.     

Adventitious presence of transgenic events in the maize supply chain in Peru: A case study

Cultivation and trade of transgenic or genetically modified organisms (GMO) and commodities has become widespread worldwide. In particular, production of transgenic crops has seen an accelerated growth along with a complex regulatory process. Current Peruvian legislation prohibits import of transgenic seeds and cultivation of transgenic crops in National territory but allows import of GMO-derived products and commodities. In addition, there is legislation that mandates the labeling of food products containing transgenic ingredients but the labeling threshold is still under discussion and the enforcement of this law is on hold. In this context, we evaluated adventitious presence of transgenic events in locally traded yellow maize using PCR- and immuno-based detection methods. Our results indicated that contamination during the distribution system of lots derived from non-transgenic maize was unavoidable and generally below 1.0% (w/w). Transgenic event MON810 was found in truck-loads of nationally grown maize. In general, frequencies of GMO-derived targets in whole-grain lots were 2.2% (GMO content ≥ 1%), 16.4% (GMO content ≤ 1%) and 81.3% (GMO content below our detection levels). When samples of de-germinated maize where evaluated, frequencies were 25.6% (GMO content > 0.9%), 65.1% (GMO content ≤ 0.9%) and 9.3% (GMO content below our detection levels). We believe this information will aid policy makers in establishing a suitable threshold for trade and product labeling as well as to conduct further investigation on other crops and scenarios.     

Development and in-house validation of a competitive ELISA for the quantitative detection of gluten in food

Wheat gluten contains peptide sequences, which activate specific T cells causing a chronic inflammation of the small intestine in celiac disease patients. It is well established that next to wheat gluten, the gluten-like proteins in barley and rye are similarly harmful to celiac disease patients whereas oat is generally considered safe. This study focuses on the development of an ELISA method for the detection of native and processed gluten proteins. The developed test utilizes a monoclonal antibody specific for the DQ2.5-glia-α3 T cell epitope present in the α-gliadins, which are part of wheat gluten that triggers celiac disease. The developed competitive ELISA uses a synthetic DQ2.5-glia-α3 peptide standard for calibration. The conversion from the measured DQ2.5-glia-α3 peptide concentration to gliadin content is achieved by using the experimentally determined multiplication factor of 250. The gluten content can be then calculated by multiplying the gliadin concentration by a factor of 2. A simple sample preparation method with 60% ethanol is used to extract the disease-causing proteins from cereals and processed foods. The assay was found to be specific for the detection of gluten from wheat, barley and rye with no cross-reaction with 8 tested oat varieties. The LOD and LOQ for gliadin were calculated based on the results obtained for 60 blank oats samples and they were 2.9 and 3.6 ppm, respectively. The assay could detect as little as 0.01% wheat gluten and gluten-like proteins from rye and barley in oats. The ELISA was also found to be applicable to the analysis of a range of processed food such as sauces, beers, soups and bread. In conclusion, the developed assay is a sensitive, specific and cost-effective tool for screening cereals and processed foods for the presence of harmful wheat gluten and gluten-like proteins from barley and rye.     

Mass spectrometry quantification of beef and pork meat in highly processed food: Application on Bolognese sauce

Food frauds have become a very important issue in the field of food quality and safety. The risk of food adulteration is higher in highly processed food and mainly affects high added value foodstuff. The methods currently available to face this issue, PCR and ELISA, are very sensitive and specific, but they have some limitations. In the present work, tandem mass spectrometry is presented as an emerging approach to detect beef and pork meat in very complex and highly processed food matrices, such as Bolognese sauce, both in qualitative than in quantitative way. The detection is achieved using two different marker peptides, specific for beef and pork meat, both deriving from α2-collagen chain. Then, a calibration curve is set up using real sauces made by different percentages of pork and beef meat in a working range from 0 to 100%. The method here developed allows to quantify beef and pork meat in a complex product such as Bolognese sauce.     

The efficacy of disinfectant misting in the lairage of a pig abattoir to reduce Salmonella and Enterobacteriaceae on pigs prior to slaughter

Water misting/showers are used in abattoir lairages to improve meat quality, and to cool and calm pigs after transport and during hot weather. One novel approach, which has not been investigated to date, is to add a disinfectant to the misting water as a means of topically reducing Salmonella on pigs prior to slaughter, thereby potentially controlling this organism in the abattoir. The objective of this study was therefore to evaluate misting with water or with Virkon^® S (an approved disinfectant for use in the presence of animals), for their ability to topically reduce Salmonella on high seroprevalence pig herds before stunning and to reduce Enterobacteriaceae.Three experimental groups were investigated: control group (i.e., no misting); water group (misting with cold, 15–17 °C, water, herein referred to as water); and a disinfectant group (misting with 0.5% Virkon^® S). As pigs entered the abattoir, each animal was swabbed along its back before being allocated to its experimental group. Each group was randomly assigned to one of 3 lairage pens that were separated by non-trial pens. After 30 min of misting with water or disinfectant, pigs were moved to the stunning area, where each pig was again swabbed, as above. Swabs were analyzed for the presence of Salmonella and enumeration of Enterobacteriaceae.Before misting, Salmonella prevalence on the pigs was 79.0%, 72.1% and 83.6% for the control, water and disinfectant groups, respectively. After misting, Salmonella prevalence increased to 94.3% in the water group; whereas for the disinfectant group, the prevalence increased marginally to 85.9%. No change in Salmonella prevalence was detected for the control group. In line with the Salmonella results, no significant differences were observed in Enterobacteriaceae counts in the control group at either time point (4.37 and 5.01 log₁₀ CFU/cm², respectively) or in the disinfectant group before and after misting (4.02 and 4.26 log₁₀ CFU/cm², respectively). However, a 2.3 log₁₀ CFU/cm² increase in Enterobacteriaceae was recorded for the water group after misting as compared to before misting (p < 0.05).Since misting with water alone increased topical Salmonella contamination on pigs before slaughter, a risk assessment based on known Salmonella data, meat quality and welfare is recommended to determine whether its use is justifiable. On the other hand, the findings from this study suggest that misting with Virkon^® S at 0.5% could have a role in topical antisepsis of pigs contaminated with Salmonella prior to slaughter and as such this warrants further investigation.