Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. An indicator based electrochemical disposable genosensor for the voltammetric detection of NOS-terminator, a genetic element present in GMOs is described as a possible substitute method for the common technique of gel electrophoresis and fluorescent image analysis. The biosensor relies on the immobilization of the 25-mer single stranded oligonucleotides (probe) related to NOS-terminator DNA sequence and the relative binding of this sequence with the polymerase chain reaction (PCR) amplified samples from certified reference material (CRM) of Roundup Ready soybean (Fluka) at a screen printed electrode (SPE). The extent of hybridization between the probe and target DNA is determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), as the hybridization indicator. The difference between the MB signals, obtained from the hybrid modified and probe modified SPEs, is used to detect GMOs from PCR amplified DNA samples. Numerous factors affecting the hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified soya and maize.     

Results of an interlaboratory assessment of a screening method of genetically modified organisms in soy beans and maize

The preliminary results on an interlaboratory trial on the detection of genetically modified organisms (GMO) are presented. The method applied is based on the detection of modified DNA using the polymerase chain reaction (PCR) for amplification. The amplified fragments analysed are derived from the 35S promotor and the NOS terminator used for modification and are present in 26 from the 28 GMOs currently already approved or under approval by the competent authorities. This method fits as a screening method indicating the presence of GMO in food. However, it does not allow an identification of the kind of GMO present in the samples. Samples of soybean and maize flour containing 0%, 0.1%, 0.5% and 2% GMO had been prepared for this study and are also already commercially available. In this paper the combined results from 27 laboratories are presented, indicating that on average the probability of false positive or false negative results is only about 1% for soybeans and below 5% for maize.     

Event-specific qualitative and quantitative PCR detection of LY038 maize in mixed samples

With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples.     

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.     

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.     

Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed

A diverse range of genetic elements has been used to develop genetically modified organisms (GMOs) over the last 18 years. Screening methods that target few elements, such as the Cauliflower Mosaic Virus 35S promoter (P-35S) and Agrobacterium tumefaciens nopaline terminator (T-nos), are not sufficient to screen GMOs. In the present study, a multiplex PCR system for all globally commercialized GM soybean events was developed to easily trace the events. For this purpose, screening elements of 24 GM soybean events were investigated and 9 screening targets were selected and divided into three individual triplex PCR systems: P-35S, ribulose-1,5-bisphosphate carboxylase small subunit promoter of Arabidopsis thaliana, T-nos, T-35S, pea E9 terminator, open reading frame 23 terminator of A. tumefaciens, proteinase inhibitor II terminator of potato, acetohydroxy acid synthase large subunit terminator of A. thaliana, and the revealed 3′ flanking sequences of DP-305423-1. The specificity of the assays was confirmed using thirteen GM soybean events as the respective positive/negative controls. The limit of detection of each multiplex set, as determined using certified reference materials of specific GM events, ranged from 0.03 to 0.5%, depending upon target. Furthermore, 26 food samples that contained soybean ingredients, which were purchased from the USA, China, Japan, and Korea, were analyzed, 17 of which contained one or more GM soybean events. These results suggest that the developed screening method can be used to efficiently track and identify 24 GM soybean events in food and feed.     

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.     

Fluorophore double stranded probe-multiplex quantitative PCR method for detecting transgenic component of promoter derived from Cauliflower Mosaic Virus and nos terminator derived from Agrobacterium tumefaciens simultaneously

The article is to establish multiplex PCR method for quantitative detecting transgenic component promoter derived from Cauliflower Mosaic Virus (CaMV 35S) and nos terminator derived from Agrobacterium tumefaciens (Tnos) in foods. According to the specific sequence of CaMV 35S and Tnos which have been used in genetically modified organisms (GMOs) frequently, and the sequence of soybean endogenous lectin gene, three pairs of primers and corresponding fluorophore double stranded probes (FDSP) were designed to allow for quantitative detecting of GMOs. FDSP designed with maximal specificity also showed the greatest detection sensitivity, and the ease in design, the simple single-dye labeling chemistry. FDSP-multiplex quantitative PCR (FDSP-MQPCR) methods were established for the detection of transgenic component CaMV 35S and Tnos simultaneously. Ten soybean flour samples were tested with FDSP-MQPCR method. The method gives five positive-samples with quantitative results in 5 h, and accuracy rate is above 97.0%. The described methods enabled a sensitive, specific, simple, and accurate detection of transgenic component and thus provide a useful tool for quantitative analysis of raw and processed food products. FDSP-MQPCR method has not only improved detection efficiency and result credibility, but also has guaranteed the better accuracy and repetitiveness.     

Real-time PCR-based detection and quantification of genetically modified maize in processed feeds commercialised in Malaysia

The present study which dealt mainly with processed feeds and some maize samples sold commercially in Malaysia evaluated the implementation of a real-time PCR cycling system for singleplex screening of eight target sequences (lectin, hmg, adh1, p35S, NK603, GA21, MON810 and MON863) and quantification of four genetically modified (GM) maize events (NK603, GA21, MON810 and MON863). The effects of using proprietary glass magnetic particles to bind DNA to their surface were also investigated in terms of DNA quantity, purity, integrity, quality and its overall effect on DNA amplification. GM material was present in 26.2% feeds and 65% maize samples. All GM samples contained MON810 followed by NK603 (47.5%), GA21 (25%) and MON863 (2.5%). Single-event and multiple-events were identified in the GM samples with 50% of the GM samples containing multiple-events. The present study which represents a fast and reliable methodology would provide an overview of the presence and levels of GMOs in feeds and maize in Malaysia.     

Development and application of DNA analytical methods for the detection of GMOs in food

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.

Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.

If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.

Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.

Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.     

Detection of banned antibacterial growth promoter in animal feed by enzyme-linked immunosorbent assay: Method validation according to the Commission Decision 2002/657/EC criteria

An enzyme-linked immunosorbent assay (ELISA) test has been applied for the qualitative screening analysis of virginiamycin and bacitracin in feedingstuffs at level of 1 mg/kg. The ELISA validation study was performed according to the Commission Decision 2002/657/EC criteria established for qualitative screening methods in food. In this regard, the following parameters were determined: detection capability (CCβ), specificity and ruggedness. The resulted CCβ values were <1 ppm for both molecules and no interferences from matrix effects were observed.Slight variations of some critical factors in the sample pre-treatment and clean up were deliberately introduced for ruggedness evaluation and they did not result in any negative effect on the detection of virginiamycin, while ruggedness evaluation on the detection of bacitracin showed some critical factors. The proposed method is suitable for qualitative screening analysis of virginiamycin and bacitracin in conformity with the current EC performance requirements.     

Detection of verocytotoxin-producing Escherichia coli (VTEC) in minced beef and raw milk by colony blot hybridization

Verocytotoxin-producing Escherichia coli (VTEC) are foodborne pathogens that cause outbreaks linked to consumption of meat and raw milk. In this note the authors report results obtained from a survey conducted on minced beef and raw bovine milk samples using a Multiplex PCR (M-PCR) for the detection of eae, stx₁, stx₂ and hlyA genes as a screening step followed by a colony blot hybridization (CBH) technique for the isolation of the VTEC. Of 100 minced beef and 123 raw milk samples, 13 (13%) and 7 (5.7%) were positive in the M-PCR and among these 9 and 3 strains were isolated using CBH, respectively. All isolates showed the presence of the stx₂ gene, single or in association with the other investigated genes. None of the isolates belonged to the O157, O26, O91, O103, O111 and O145 serogroups. The study showed that the use of M-PCR for the screening of samples coupled with a sensitive and specific detection technique, could improve the possibility of detection of VTEC strains in foods. Moreover, the presence of VTEC in minced beef and in raw milk confirms their important role as putative vehicles of infection to humans. Stringent control of these foodstuffs is essential for food safety purposes.     

Detection methods for genetically modified crops

Due to the market introduction of genetically modified crops (GMOs) as the Roundup Ready (RR) soya and Bt corn, the European food industry came face to face with the question of the use and labeling requirements on GMO crops and its derivatives. Although even today, no defined European legislation is available, a definitive need for detection methods exists. Both DNA and protein based methods have been developed and applied for the detection of RR soya beans and its derivatives. For the CP4 synthase, synthetic peptides corresponding with the antigenic and non-homologous parts of the CP4 synthase were synthesized and mono-specific anti-CP4 synthase monoclonal antibodies were prepared by hybridoma technology. The monoclonal antibodies were able to detect the CP4 synthase in the RR soya using Western blotting analysis. Detection limits were found between 0.5% and 1%. The method is currently validated for half-and final products. The applied DNA methodology was making use of polymerase chain reactions (PCR) using sets of primers along the gene encoding the Agrobacterium CP4 synthase. DNA extraction and purification conditions were examined on a case-by-case approach for a scala of soya products (lecithin, oil, soybean meal, soy protein isolates etc.), half-products and final consumer products. Detection limits were found between 0.01% and 0.1%. In this paper a comparison will be made between the two types of methods in relation to sample preparation, sensitivity, validation and the use for half-products and final consumer products.     

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.     

PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products

Alternaria spp. contamination and subsequent production of mycotoxins is a common problem in vegetable crops. Identification of Alternaria species by traditional methods requires specific skills and may not detect toxigenic moulds inactivated by food processing. By using molecular methods such as PCR the detection of Alternaria spp. becomes possible directly from the food or feed samples. In this study, a PCR method based on the Internal Transcribed Spacer (ITS) genetic marker has been used for detection of Alternaria spp. in raw and processed commercial tomato samples. Occurrence of altenuene, alternariol and alternariol methyl ether in the samples was analysed by high-performance liquid chromatography (HPLC) in order to assess the ability of the PCR assay to identify tomato samples containing Alternaria mycotoxins. The PCR assay revealed the presence of Alternaria spp. DNA in 41 out of 90 commercial samples (45.6%), while HPLC detected at least one of the Alternaria mycotoxins within 31 of the PCR positive samples. Detection of Alternaria DNA correlated well with the presence of the analysed Alternaria mycotoxins, indicating that the PCR protocol developed in this work for detection of Alternaria spp. DNA could be used as an indirect marker of the presence of Alternaria mycotoxins in raw and processed tomato products.     

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

Real-time PCR assay for detection of Trichinella in meat

Trichinella are a group of widely distributed parasites which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked food. A TaqMan^®-LNA probe Real-time PCR assay targeting the 5S rRNA was developed allowing the simultaneous detection of the 10 species and 3 genotypes of Trichinella present in meat tissues. The detection limit employing dilutions of genomic DNA was 2 pg and the determination of the detection limt in terms of ppm was 1 ppm.The main novelty of this work lies in the fact that it can assure the absence of 10 species and 3 genotypes of Trichinella in an only assay. The proposed methodology is rapid, robust, highly sensitive and readily adaptable in routine molecular diagnostic laboratories, and can be employed as molecular screening method in order to assess the food security.