An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples

The purpose of this study is to establish a reliable detection method specific for event 98140, a type of multiple herbicide-resistant corn. Based on the 3′- flanking sequence of event 98140, qualitative and quantitative PCR detection assays were developed. The results revealed the LOD of 0.05% of GM maize for qualitative PCR assay and 4 transgenic haploid genome copies for quantitative PCR assay used in this study. The LOQ was 20 transgenic haploid genome copies, and based on this, as low as 0.05% of 98140 genomic DNA could be accurately and quantitatively detected by means of the quantitative method. Two mixed corn samples, with known 98140 contents (2% and 0.5%), were used to verify the developed real-time PCR system, and the expected results were observed. The results indicated that the developed event-specific PCR methods could be used for the identification and quantification of maize line 98140.     

Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 5′ and 3′ flanking sequences

Plasmid molecule based reference material (RM) has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification, while most of the currently developed plasmid RM can only be used for one specific target detection. In this study, a flexible plasmid RM pNK containing three DNA fragments, i.e. 5′ and 3′ event-specific sequences of maize NK603 and endogenous gene zSSIIb, was developed. We have proved that pNK is suitable for using as a calibrator in both 5′ and 3′ event-specific detection of maize NK603, compared with that of genuine genomic DNA. The limit of detection (LOD) was 10 copies of pNK DNA in conventional PCR assays. The absolute LOD and limit of quantification (LOQ) in quantitative PCR assays were 5 and 25 copies. The standard curves targeting to zSSIIb, 5′ and 3′ event-specific sequences based on pNK DNA showed high reaction efficiency and good linearity. Also, low bias and variations were obtained in practical samples quantification using pNK as the calibrator. These results demonstrated that the developed pNK is flexible and suitable for identification and quantification of maize NK603, as a preferable substitute of RM from the plant raw material.     

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.     

Development of an event-specific assay for the qualitative and quantitative detection of the genetically modified flax CDC Triffid (FP967)

The genetically modified flax, event FP967, with tolerance to sulfonylurea herbicides, is one of the commercial genetically modified events approved in Canada and USA.This event is not authorized in Switzerland and EU, therefore, a method to specifically detect the CDC Triffid line, was required. We revealed the 3′ integration junction sequence between host plant DNA and the integrated gene construct FP967 by means of Restriction Site PCR and both a qualitative and a quantitative PCR detection assays were developed. The qualitative PCR revealed a limit of detection of 0.01% of GM flax in 100 ng of genomic DNA. The quantitative PCR assay showed a limit of detection of about 9 haploid genome copies. The specificity and sensitivity of the assay indicate that the developed event-specific PCR methods can be used for identification and quantification of FP967 flax.     

Applicability assessment of a real-time PCR assay for the specific detection of bovine,ovine and caprine material in feedstuffs

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of bovine, ovine and caprine processed animal protein (PAP) in industrial feedstuffs. The method uses species-specific primers and probes targeting short mitochondrial D-loop sequences, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 126 industrial feed samples that were manufactured to reproduce rendering processes of commercial feeds destined for farmed animals. The assay successfully classified samples as positive or negative according to the ruminant composition, enabling qualitative detection of banned material in feeds at levels as low as 0.1%. Although the method provides quantitative potential, results suggest that the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatments of the feeds, which affect the amount and quality of amplifiable DNA.     

Development of a multiplex PCR method for testing six GM soybean events

Genetically modified (GM) soybean and derived products make up a large part of the biotech-derived food and feed market. As more GM soybean varieties have been approved for commercialization, labeling requirement by South Korea and other countries needs the technical testing methods. This paper reports the development of a multiplex PCR method for identifying six commercialized GM soybean events using the event-specific fragment. Event specific primers targeting Roundup Ready Soybean (RRS, GTS40-3-2), A2704-12, DP356043-5, MON89788, A5547-127, and DP305423-1 were designed, and a multiplex PCR assay consisting of six event-specific fragments and one endogenous lectin fragment was developed. The specificity of the event-specific PCR method was confirmed using 20 GM events of maize, soybean, cotton, and canola. The limit of detection (LOD) for each event in the multiplex PCR is approximately 0.05%. Intra-lab validation by two different operators confirmed the specificity and LOD of this multiplex PCR method. The method was used to test 30 soybean-derived foods from South Korean and US markets, and results revealed three varieties of GM soybean (RRS, A2704-12, and MON89788) in 19 of the 30 food samples tested. This work provides an efficient and cost-effective approach for event-specific analysis of six commercialized GM soybean varieties and related processed foods in Korea.     

Multiplex real-time PCR assays for simultaneous detection of maize MON810 and GA21 in food samples

This work describes a quantitative multiplex real-time PCR method optimized for the detection of maize MON810 and GA21. The use of specific primers and of labeled probes by real-time PCR allowed for the simultaneous detection and confirmation of amplicon identity and increased the reliability of the technique and the number of PCR applications to food analysis.Two different endogenous genes, Zein and Adh1, were evaluated for quantitative use as accountable for continuous development of maize traits. The quantification is based on a calibration standard curve obtained with the DNA extracted from Certified Reference Materials (CRMs). The limit of detection (LOD) and limit of quantification (LOQ) of the triplex assays developed was set at 3 and 36 copy numbers respectively.     

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.     

Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick

LAMP–LFD, loop-mediated isothermal amplification (LAMP) combined with a lateral-flow dipstick (LFD), was developed and evaluated as a new method for the detection of Roundup Ready soybean (RRS). Biotinylated LAMP amplicons were produced by two sets of six designed primers that specifically recognized the endogenous gene (Lec1) and the event-specific 5′ -junction region (G35S) of RRS followed by hybridization with FITC-labeled probes and LFD detection. The following optimized conditions for the LAMP assay were used: deoxynucleotide triphosphate (dNTP) concentrations ranging from 0.6 to 3.2 mM, 6 mM Mg²⁺, 4 U Bst DNA polymerase and a 1:6 ratio of outer to inner primers. The LAMP–LFD results were generated within 50 min. The detection limit of LAMP–LFD was 2.4 copies of the linearized plasmid pTLH10 and was 20 times more sensitive than conventional PCR. We demonstrated the high specificity of LAMP–LFD by testing processed soybean products, genetically modified (GM) maize and Bt-cotton meal. The novel LAMP–LFD setup presented here is simple, rapid, and has the potential for future use in the detection of GM ingredients in feed and food products.     

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.     

Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Development of a real time PCR assay for detection of allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitized individuals. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a TaqMan real-time PCR method for specific detection of peanut in commercial food products. The assay uses two genetic makers in the Arah2 (125 bp) and ITS1 (90 bp) gene respectively, and TaqMan probes. The nuclear 18S rRNA gene was employed as a positive amplification control. Results obtained on sensitivity of both Arah2 and ITS primers with mixtures spiked with different concentrations of the target showed detection levels of 10 and 0.1 ppm, respectively. The applicability of the real-time PCR protocol to detect the presence of peanut DNA in commercial food products was determined through analysis of 123 different commercial products with the ITS primers which aimed higher sensitivity than Arah2 primer pair. The performance of the real-time PCR proposed herein allows a highly sensitive detection of peanut DNA in all the different food products, which declared to contain peanut material as well as in others were the presence of peanut or its traces wasn't declared in the labeling.     

Monitoring the occurrence of genetically modified soybean and maize in cultivated fields and along the transportation routes of the Incheon Port in South Korea

In South Korea, imported genetically modified (GM) soybean and maize have been approved for both human consumption and use in animal feed, but not for use in cultivation in fields. This study was conducted to survey the spread of GM soybean and maize in South Korea using multiplex-PCR analysis methods. Cultivated soybean, wild soybean, and maize leaf samples were collected from 26 major areas of soybean cultivation throughout eight provinces. Roadside areas near a major grain port in Incheon were also surveyed to investigate the escape and spread of GM seeds and plants. Amplification results showed that no GM soybean or maize was collected from cultivated fields. However, four GM maize plants were found in samples collected from the roadside near a grain transporting company at the Incheon Port. Based on PCR analysis using GM maize event-specific primers, it was suggested that a maize plant may be Mon810, while the other plants may be stacked events: Mon863 × Mon810 or Mon88017 × Mon810.     

MDEA溶液中无机阴离子的定性及定量分析方法研究

建立了使用离子色谱对MDEA溶液中无机阴离子进行定性与定量测定的方法。通过对不同的淋洗液模式（等度模式、一步梯度模式、多步梯度模式）进行研究比较,确定了分离度最好模式为多步梯度模式。采用多步梯度洗脱方式分别对F-,Cl-,Br-,NO3-,SO42-无机阴离子的混合标准溶液系列进行测定,建立了外标定量曲线, 5种无机阴离子的质量浓度在其线性范围内的相关系数可以达到99%以上,检出限可达到10 μg/L以下,该方法在线性范围内线性度较好,检测灵敏度高。重复性试验和加标回收试验结果表明,该方法的相对标准偏差小于5%,加标回收率在95.9%～105.6%之间,准确度较高。通过对使用了一年左右的MDEA溶液进行测试,可知溶液中含有Cl-,NO3-,SO42- 3种无机阴离子,其质量浓度分别为1191.055,3.155,41.595 mg/L。     

Real-time PCR for quantitative meat species testing

A method for quantitative meat speciation is described which combines the use of real-time PCR with species specific and ‘universal’ primers to measure individual species content and total meat content respectively. A comparison of the cycle number at which universal and species specific PCR products are first detected, in combination with the use of reference standards of known species content, is used as the basis for determining the percentage of a given species in a mixed sample. Importantly, the use of universal primers allows differences in DNA quality between samples and reference standards to be taken into account, while the use of real-time PCR allows measurement at an early stage in the PCR process which is inherently more accurate than the end point analysis associated with gel-based systems. This paper describes the quantification of beef in mixed samples to illustrate the principle of this approach.     

Quantitative detection of goats’ milk in sheep’s milk by real-time PCR

The need to support food-labeling legislation has provided a driving force for development of analytical techniques for the analysis of food ingredients. In this study, the development of a method for quantification of goats’ milk in sheep’s milk mixtures is described. The technique involves the use of a real time PCR technique, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA). The method combines the use of goat-specific primers that amplify a 171 bp fragment from goat DNA, and mammalian-specific primers amplifying a 119 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan) that hybridizes in the “goat-specific” and also in the “mammalian” DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C_t) at which mammalian and goat-specific PCR products are first detected, in combination with the use of reference standards of known caprine content, allows the determination of the percentage of goats’ milk in a milk mixture. The assay was used to analyze raw and heat-treated milk binary mixtures (goat/sheep), enabling the quantification of goats’ milk in the range 0.6–10%. The reported PCR assay may represent a rapid and straightforward tool applicable to the authentication of milk and other dairy products.     

PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins

The real-time PCR-based system has been developed for the detection and quantification of the most common Fusarium species with the similar profiles of mycotoxins they can potentially produce. Corresponding group-specific primers for various Fusarium fungi have been designed based on the translation elongation factor 1α (tef1α) gene sequences. The specificity of the selected primers and TaqMan probes has been tested with 26 single-spore isolates of seven most common Fusarium species and 21 wheat and barley grain samples. PCR results were analyzed relative to the most common mycotoxin deoxynivalenol (DON) content in infected grain samples.     

Monitoring the occurrence of genetically modified maize at a grain receiving port and along transportation routes in the Republic of Korea

The cultivation area of genetically modified (GM) crops is increasing all over the world. Though no land in the Republic of Korea is currently used for the cultivation of GM crops, GM crop imports for food and foraging purposes are continuously increasing. This may promote the unintentional escape of GM crops. This study was conducted to investigate whether imported GM maize is released into our environment during the transportation of grain in the Republic of Korea. Based on PCR analysis, most of the maize grains in the forage products were GM, and about 50% of the grains were germinated. Monitoring was conducted in two major grain receiving ports, 15 feed manufacturing plants, and 14 livestock barns in five provinces of the Republic of Korea from July to September 2007. We found many spilled maize grains around open storage areas of ports and along truck transportation routes near feed manufacturing plants. Established maize plants were not found at or around Incheon port. However, we found 18 established maize plants at the Gunsan port, 15 of which were GM. We also found eight GM maize plants around four feed manufacturing plants and in two livestock barns. Based on the event-specific PCR analysis, three maize events (NK603, Mon810, and TC1507) were identified. Though several GM maize plants were found around the port and feed manufacturing plants, most of these facilities were located inside the industrial park and were far from cultivated fields, likely rendering the impact of these GM maize on the natural environments negligible. However, most of the livestock barns were close to cultivated areas. Moreover, maize plants were cultivated for food or feed near some livestock barns. This practice may facilitate gene flow from GM maize to non-GM maize plants. Therefore, continuous monitoring is necessary to detect the occurrence of GM maize, and appropriate action should be taken to prevent genetic admixture in our environment.     

A multiplex PCR assay for fraud identification of deer products

Attempts were made to established one-step multiplex PCR assay for the fraud identification of the mostly used species in deer products (bovine, ovine, porcine and poultry). Primers were selected from published papers or designed in the well-conserved region of tRNA-Val and 16S rRNA mitochondrial genes after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 124, 183, 225 and 290 bp length for bovine, poultry, ovine and porcine, respectively. The detection limit was 1 ng for porcine and ovine primers, 5 ng for poultry primers and 0.5 ng for bovine primers. The results demonstrated that fraud phenomena are very epidemic in the deer products, especially heart, blood, penis and antler products.The multiplex PCR described in this study, proved to be very sensitive and reliable in species identification, could be considered as a further improvement of traditional methods based assay for the identification of deer products.