Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods should preferably enable detection of several different GMOs. Here we present a simple, robust, qualitative, nineplex PCR method for event-specific detection of maize T25, GA21, TC1507, MON863, MON810, NK603, construct specific detection of BT176, BT11 and detection of the endogenous hmga maize reference gene. PCR is carried out with primers labelled with fluorescent groups and the amplicons are detected using fluorescence capillary electrophoresis. Using mixtures of DNA from different certified reference materials, the detection limit was determined to approximately 0.1% for each GMO. Good agreement was observed in 85 of 88 determinations when eleven food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. Discrepancies were only observed for one GMO at or close to the detection limit. The presented method is therefore suitable for screening purposes for food and feed containing the most common maize GMOs.     

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.     

Parallelised real-time PCR for identification of maize GMO events

Identification of specific material derived from genetically modified organisms (GMO) present in food, feed or seed samples screened positive for the presence of genetic modification(s) is mandatory for the official food and feed control in the European Union. Since the introduction of regulation (EC) No 1829/2003 in 2004, the number of maize GMO events either approved in the EU or with a pending application grew constantly. By the sheer multitude of events and crossed events (stacks), maize poses a special challenge on official food and feed control. We developed a modular qualitative detection system for the parallel identification of maize GMO events to cope with the increasing number of GMO potentially present in routine samples. This system is based on validated real-time PCR assays in a microtitre plate format grouped modularly by crop species. The maize module identifies in parallel, i.e. simultaneously, 15 maize events and RoundupReady soy in a single analytical run of approximately 2 h. Maize modules can be conveniently prepared in advance and stored at −20 °C until use. Ready-to-use reference DNA mixtures serve as positive controls. The modular approach is flexible as it allows easy change or addition of individual detection reactions, if necessary, e.g. when new validated methods become available. 23 food, 14 feed and 8 seed samples were successfully analysed with the maize module. The parallel detection of nine different GMO maize and soy events in single routine samples demonstrated the usefulness of the parallelised modular approach for routine GMO analysis.     

Detection of GM Canola MS11, DP-073496-4, and MON88302 events using multiplex PCR coupled with capillary electrophoresis

As of 2020, 11 GM canola events have been authorized as food for humans in Korea. However, there are no simultaneous multiplex detection methods for 3 GM canola events (DP-073496-4, MON88302, and MS11). Thus, we established the multiplex polymerase chain reaction (PCR) method coupled with capillary electrophoresis to detect 3 GM canola events. To verify the specificity of event-specific primers, various GM crops of 3 GM soybean events, 6 GM maize events, 2 GM cotton events and 11 GM canola events were prepared. The limit of detection of the developed multiplex PCR was approximately 0.0125% for 3 GM canola events. Certified GM canola and stacked events were analyzed to validate the developed multiplex PCR. This study focuses on establishing multiplex PCR coupled with capillary electrophoresis for newly approved GM canola events and contributes to efficient monitoring GM canola samples in Korea.     

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.     

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 moniliformin in maize using capillary zone electrophoresis.

Moniliformin is a mycotoxin produced by certain fungi pathogenic to maize. It is capable of causing disease in domestic animals, possibly through inhibition of pyruvate dehydrogenase. Testing for MON commonly involves extraction of maize, isolation of moniliformin using solid-phase extraction columns and detection with high-performance liquid chromatography (HPLC) or gas chromatography. A capillary zone electrophoresis-diode array detection (CZE-DAD) method for determination of moniliformin in maize is reported. The extraction and isolation procedures are similar to those of a commonly used HPLC method, while the detection step requires only 10 min. Sixty-three samples of maize were tested by an established HPLC method using absorbance at 229 nm (HPLC-ultraviolet light) and by the CZE-DAD method. The limit of detection of the CZE-DAD method was 0.1 microg MON g(-1) maize compared with 0.05 microg g(-1) for the HPLC-ultraviolet light method. The CZE-DAD method gave good agreement with the HPLC-ultraviolet light method for samples tested at levels up to 1500 microg g(-1), with a linear regression of r(2) = 0.996.     

Detection of Clostridium botulinum neurotoxin coding genes: analysis of PCR products by real time versus capillary gel electrophoresis methods

In this work, two PCR-based methods have been developed for the detection of Clostridium botulinum strains carrying the gene coding for C. botulinum neurotoxin C (BoNTC) responsible for avian botulism. Both methods are based on the same amplification primers designed using multiple sequence alignments between toxin C coding sequences from DNA sequence databases. The first is a real-time PCR method, using a Taqman-MGB probe. The second uses conventional end-point PCR, followed by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). A comparison between both methods has been established for the individual and simultaneous detection of toxin C (BONTC) or bacterial 16S (BACT) sequences from C. botulinum. The results indicate that, in general, the same sensitivity was achieved by using RT-PCR and PCR-CGE-LIF allowing the detection of both C. botulinum amplicons from concentrations as low as 7 × 10⁻⁵ μg/ml of total genomic DNA. Some other features from RT-PCR and CGE-LIF are also critically discussed in this work, including quantification capability, size determination, analysis speed and identification strategies, to provide enough information to adequately select the best analytical technique in each case. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

烟草五种土传病原菌的多重PCR快速检测

【目的】建立一种可同时检测烟草黑胫病菌（Phytophthora parasitica）、青枯病菌（Ralstonia solanacearum）、立枯病菌（Rhizoctonia solani）、根腐病菌（Fusarium oxysporum）和根黑腐病菌(Thielaviopsis basicola)5种烟草重要土传病原菌的五重PCR快速检测方法。【方法】筛选5种病原菌的特异性引物组合,通过不同的引物浓度和退火温度对多重PCR体系进行优化,检测体系的灵敏度,并对土壤和植株样品进行测试,验证其实用性。【结果】根据青枯病菌fliC基因、立枯病菌RPB2基因、根腐病菌COI基因以及根黑腐病菌TEF1基因设计特异性引物,并结合已报道的黑胫病菌parA1基因的特异性引物,成功建立5种烟草土传病害的多重PCR检测方法。反应体系(25μL):Sf1/Sr1、Ff1/Fr1、Pf1/Pr1每条引物分别0.3μL,Rf1/Rr1每条引物各1.8μL,TBf1/TBr1每条引物各1μL,2×PCR Mix 12.5μL,退火温度为58℃,灵敏度可达到100 pg/μL。【结论】本研究建立的多重PCR体...     

Fast analysis of proteins in wines by capillary gel electrophoresis

Wine proteins play an important role in different characteristics of wine (e.g., aroma and body, foaming in sparkling wines). They can also cause a number of technological problems during vinification and may be responsible for the appearance of turbidity in bottled wine. These important features of proteins in wine have made necessary the development of new and fast analytical methods that can provide deeper knowledge about these biopolymers. However, separation and characterization of wine proteins is difficult and time-consuming mainly due to their low concentration and large number of interfering compounds. Besides, long sample preparation protocols can bring about protein decomposition. This paper proposes a new and fast method for carrying out the analysis of the protein fraction of wines. The procedure consists of direct treatment of wine using a centrifugal filter device (CFD), denaturation of the proteinaceous fraction with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and subsequent CGE analysis of SDS-proteins. Results on the molecular weight (Mw) and relative quantity of proteins of wines are attained in about 1 h with this procedure. The method is applied to analyze different wines from Canary Islands. To our knowledge, this is the first report of separation of wine proteins according to their Mw by CGE.     

采用毛细管凝胶电泳技术检测蜂王浆新鲜度

新鲜度是评价蜂王浆产品质量一个重要指标。文中采用毛细管凝胶电泳技术对蜂王浆水溶性蛋白质进行了分离测定。结果表明:毛细管电泳技术对蜂王浆水溶性蛋白定量准确、重复性、精密度、稳定性各项指标良好,操作简便,快速,适合于蜂王浆产品的新鲜度的评价。为了筛选新鲜度指示蛋白,将王浆样品在不同温度下放置不同时间,分别进行毛细管电泳分析,最终从蜂王浆19个蛋白峰中筛选到2个蛋白峰,含量随存储温度和时间呈现规律性变化,可以作为蜂王浆新鲜度指示蛋白。     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。     

MPCR方法检测食品中的伤寒沙门氏菌、金黄色葡萄球菌和单增李斯特氏菌

为建立一种同时检测食品中的伤寒沙门氏菌(ST)、金黄色葡萄球菌(SA)和单增李斯特氏菌(LM)的快速检测方法,分别针对伤寒沙门氏菌的鞭毛抗原基因H1-d、金黄色葡萄球菌的耐热核酸酶基因nuc、单增李斯特氏菌溶血素O上的hlyA基因设计引物,进行特异性和灵敏性实验,结果表明3条特异性扩增片段分别为458bp、279bp和243bp,经DNA测序证明其序列与模板被扩增片段一致。该方法操作简便、快速,具有良好的灵敏性和特异性。     

Detection of allergenic substances in foods by a multiplex PCR method

A multiplex PCR (M-PCR) method was developed for the detection of DNAs of plant and three allergenic substances (wheat, buckwheat, and peanut) in foods. Genomic DNAs were extracted from allergenic substances with a commercial ion-exchange type kit. Four primer pairs suitable for the specific detection of plant DNA were designed to establish a M-PCR method detecting simultaneously the specific DNAs of plant and allergenic substances. Our four designed primer pairs and the primer pair described in the Japanese official method were applied to the specific detection of plant DNA. A primer pair of Plant01-5' and Plant01-3' (amplicon size; 161 bp) was the most suitable for the specific detection of plant DNA. M-PCR was performed to detect the specific DNAs of allergenic substances using four primer pairs, a pair of Plant01-5' and Plant01-3', and three pairs for allergenic components described in the Japanese official method. The four specific PCR bands were simultaneously amplified from genomic DNAs of allergenic substances. The proposed method is simple, rapid and inexpensive.     

Detection and identification of transgenic virus resistant papaya and squash by multiplex PCR

Two separate multiplex PCR assays were developed for the detection of transgenic papaya and transgenic squash. Papaya line 55-1 contains a genetic insertion consisting of the coat protein gene of Papaya ringspot virus strain p and is resistant to infection by this virus. A multiplex PCR was developed to specifically amplify the papaya ringspot virus coat protein transgene construct and an endogenous papaya gene sequence. A third primer set was designed to amplify the -glucuronidase gene construct, which can distinguish between the commercial and noncommercial papaya lines 55-1 and 63-1. Squash line ZW-20 contains genetic insertions of the coat protein genes from Watermelon mosaic virus II and Zucchini yellow mosaic virus and is resistant to these two viruses. Squash line CZW-3 is similar to ZW-20 but additionally confers resistance to Cucumber mosaic virus. A second multiplex assay was developed to detect and differentiate squash lines ZW-20 and CZW-3 in a single reaction.     

The simultaneous separation and determination of five organic acids in food by capillary electrophoresis

The simultaneous separation of quinic acid, anisic acid, salicylic acid, benzoic acid and sorbic acid was performed by capillary electrophoresis with 20 mmol L⁻¹ NaOH–20 mmol L⁻¹ H₃BO₃ (pH 8.8) as the background electrolyte. The choices of the background electrolytes and the applied voltage were optimized. The effects of tetrabutylammonium bromide and sodium dodecylsulfate on the separation were investigated in detail. Under the optimum condition, the linearity, reproducibility and detection limit of five organic acids were shown, respectively. As an application of the method proposed, 10 samples of soy sauce and vinegar were analysed to determine benzoic acid and sorbic acid. The procedure described provides the advantages of good selectivity, rapid speed and simplicity for the separation and determination of organic acids.     

Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR

The objective of this study was to develop a rapid multiplex PCR (m-PCR) method using pure bacterial cultures and pork that would allow the simultaneous detection of five major foodborne pathogens likely to be found in pork (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and Yersinia enterocolitica). Five pairs of primers were designed to identify invA gene, hlyA gene, rfbE gene, nuc gene and ail gene for Salmonella, L. monocytogenes, E. coli O157:H7, S. aureus and Y. enterocolitica, respectively. On the basis of determining specificity of the m-PCR, sensitivity was performed with an overnight enrichment step in culture media and pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The results suggested that the m-PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. After overnight culture, the detection limit was 10³ cfu/mL for the simultaneous detection of the five target pathogens and less than 10 cfu/mL for detection of a single pathogen. The m-PCR protocol successfully detected all five organisms inoculated overnight together on pork. Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli O157:H7 were detected at levels of 142, 51, 9, 33 and 670 cfu/mL, respectively, in the pork. The m-PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.     

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.     

Detection of cow milk in goat milk by polyacrylamide gel electrophoresis

Pasteurized goat milk was adulterated with increasing proportions of cow milk and submitted to polyacrylamide gel electrophoresis. A frontal band, missing from the pattern of genuine goat milk and possessing the same electrophoretic mobility as bovine alpha S1-casein, was expressed. The area of this zone was directly proportional to the amount of cow milk added to the goat milk.