Assessment of DNA degradation induced by thermal and UV radiation processing: Implications for quantification of genetically modified organisms

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO’s) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.     

Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid,Plant‐Derived Foods

Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, C_t values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf‐life. This indicated that the combination of lower temperature thermal processes (hot‐fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced C_t values which had high correlation to time‐temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time‐temperature treatments to C_t values. Lab‐scale pasteurization studies using C_t values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant‐derived products.     

Innovative Application of Fluorescent Microsphere Based Assay for Multiple GMO Detection

An innovative multiple screening protocol allowing the detection of specific DNA sequences of p35S and epsps in GM soya flours simultaneously was developed. For the application of the Luminex xMAP technology, two different sets of fluorescent beads were cross-linked to the specific oligonucleotide probes previously amplified and labeled by polymerase chain reaction (PCR) in the presence of a biotinylated nucleotide. The detection of the amplification products bounded by the streptavidin–phycoeritrin conjugate was performed using the Luminex-100 instrument. The system allows identification of extremely low amounts of labeled targets as of 7.8 10⁻⁴ nmol of p35S antiprobe with an associated value of repeatability relative standard deviation (RSD_r) equal to 2.8. Parameters such as repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ) were considered to evaluate the performances of the assay. The potentiality of the system described in this paper enables us to look forward to a multiple target assay that is able to simultaneously detect and eventually quantify tens of target sequences occurring within the same sample preparation. The present study describes the first application of a multitarget fluorescent microsphere-based assay for DNA genetically modified organism (GMO) detection.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

A model of non‐isothermal degradation of nutrients,pigments and enzymes

Published isothermal degradation curves for chlorophyll A and thiamine in the range 100–150 °C and the inactivation curves of polyphenol oxidase (PPO) in the range 50–80 °C could be described by the model C(t)/C₀ = exp[?b(T)tⁿ] where C(t) and C₀ are the momentary and initial concentrations, respectively, b(T) a temperature dependent ‘rate parameter’ and n, a constant. This suggested that the temporal degradation/inactivation events of all three had a Weibull distribution with a practically constant shape factor. The temperature dependence of the ‘rate parameter’ could be described by the log logistic model, b(T) = log_e[1 + exp[k(T ? T_c)], where T_c is a marker of the temperature level where the degradation/inactivation occurs at a significant rate and k the steepness of the b(T) increase once this temperature range has been exceeded. These two models were combined to produce a non‐isothermal degradation/inactivation model, similar to one recently developed for microbial inactivation. It is based on the assumption that the local slope of the non‐isothermal decay curve, ie the momentary decay rate, is the slope of the isothermal curve at the momentary temperature at a time that corresponds to the momentary concentration of the still intact or active molecules. This model, in the form of a differential equation, was solved numerically to produce degradation/inactivation curves under temperature profiles that included heating and cooling and oscillating temperatures. Such simulations can be used to assess the impact of planned commercial heat processes on the stability of compounds of nutritional and quality concerns and the efficacy of methods to inactivate enzymes. Simulated decay curves on which a random noise was superimposed were used to demonstrate that the degradation/inactivation parameters, k and T_c, can be calculated directly from non‐isothermal decay curves, provided that the validity of the Weibullian and log logistic models and the constancy of the shape factor n could be assumed. Copyright © 2004 Society of Chemical Industry     

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X ₀) and the content of GM DNA in tested material (X _SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.     

Differences in quality and quantity of transgenic component in Roundup Ready soybean during bean curd preparation

Using 2.5% (w/w) transgenic Roundup Ready soybean as raw material, the effects of critical processing procedures on the fragment sizes of endogenous gene lectin and transgenic gene epsps and the quantity of transgenic component during bean curd preparation were analysed. The results showed that the different processing procedures differed in their impact on the degradation of endogenous and transgenic genes and that the transgenic content of the intermediate products and final product was consequently changed. Physical mechanical action such as jordaning caused both endogenous and transgenic genes to degrade but had more effect on the latter. The jordaning process degraded the DNA of endogenous gene from 1883 to 836 bp and that of transgenic gene from 1512 to 408 bp. The adding CaSO₄ process, predominantly a chemical reaction, had less influence on the transgenic gene than on the endogenous gene, the latter being degraded from 836 to 407 bp. The extrusion moulding process resulted in a significant degradation of DNA in transgenic gene from 408 to 190 bp but had no obvious effect on the endogenous gene. After the critical processing procedures of jordaning, syruping, adding CaSO₄ and extrusion moulding during bean curd preparation the transgenic content of the intermediate products and final product was 1.656, 0.435, 1.150 and 0.797% respectively. Copyright © 2007 Society of Chemical Industry     

Evaluation of real-time PCR results at the limit of detection

Real-time polymerase chain reaction (real-time PCR) facilitates to detect DNA fragments at very low copy numbers. Positive results, e.g. for unauthorized genetically modified organism (GMO) contamination or for allergens, may raise safety concerns and have far-reaching consequences. However, in case of very low concentrations of DNA samples, results for the same product lot or even for identical samples from different laboratories may differ. Therefore, an approach for a standardized interpretation and reporting of results obtained by real-time PCR at the limit of detection (LOD) is proposed. A quality control DNA sample containing the target at the LOD (95?%) is analysed in parallel with the real DNA sample and the respective C _T values are compared. In addition, practical approaches for in house and precision-based estimation of the LOD are presented. The proposed approach may also contribute to the current discussion on implementing a technical solution to handle DNA traces in specimen, e.g. for the detection of unauthorized GMO.     

Tracing transgenic maize as affected by breadmaking process and raw material for the production of a traditional maize bread, broa

Broa is a maize bread highly consumed and appreciated, especially in the north and central zones of Portugal. In the manufacturing of broa, maize flour and maize semolina might be used, besides other cereals such as wheat and rye. Considering the needs for genetically modified organism (GMO) traceability in highly processed foods, the aim of this work was to assess DNA degradation, DNA amplification and GMO quantification along breadmaking process of broa. DNA degradation was noticed by its decrease of integrity after dough baking and in all parts of bread sampling. The PCR amplification results of extracted DNA from the three distinct maize breads (broa 1, 2 and 3) showed that sequences for maize invertase gene and for events MON810 and TC1507 were easily detected with strong products. Real-time PCR revealed that quantification of GMO was feasible in the three different breads and that sampling location of baked bread might have a limited influence since the average quantitative results of both events after baking were very close to the actual values in the case of broa 1 (prepared with maize semolina). In the other two maize breads subjected to the same baking treatment, the contents of MON810 maize were considerably underestimated, leading to the conclusion that heat-processing was not the responsible parameter for that distortion, but the size of particle and mechanical processing of raw maize play also a major role in GMO quantification.     

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T _m-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.     

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.     

Rates of degradation of plant cell walls measured with a commercial cellulase preparation

The relationship between the degradability, determined with a commercial cellulase preparation, of the cell walls of various plant parts of Italian ryegrass, maize and red clover can be expressed as Y = A - Be^?k₁^t-Cek₂^t, where Y = percentage of cell walls degraded, t = reaction time, k₁ and k₂ are rates of degradation, and A, B, and C are constants where A = B + C. Degradability of the cell walls of Italian ryegrass or maize could be predicted accurately from the absorbance of the filtrate at λ_max 282-288 or 310-324 nm. Treatment of cell walls of barley straw with 0.1 or 1M sodium hydroxide for 7 or 20 h degraded between 12 and 41% of the walls and led to the release of p-coumaric and ferulic acids, the amount increasing with concentration of alkali and treatment time; the less concentrated alkali released more ferulic than p-coumaric acid. Treatment with the cellulase preparation of the residues from alkali treatment showed that they were almost twice as degradable as the untreated walls.     

The effect of decanoic,dodecanoic and tetradecanoic acids on silage fermentation

The ability of decanoic (C₁₀), dodecanoic (C₁₂) and tetradecanoic (C₁₄) acids to restrict the fermentation of ryegrass and lucerne was investigated with laboratory silos. With ryegrass, addition of the C₁₂ acid alone at 1 kg t^?1 of fresh crop had no effect on extent or pattern of fermentation compared to that of the untreated crop, though at 2 kg t^?1 the acid appeared to reduce the content of butyric acid but not ammonia-N in the silage. Formic acid, added alone or with C₁₀, C₁₂ and C₁₄ acids restricted extent of fermentation and butyric acid production, but in this experiment addition of these acids had relatively little effect on the content of ammonia-N in the silages. With lucerne, extent of fermentation was similar for all treatments, though the untreated silage and those which received C₁₂ or formic acid alone at 1 kg t^?1 or 1 kg t^?1 of C₁₄ acid with 1 kg t^?1 of formic acid contained butyric acid in excess of 3% of the dry matter (DM). It is concluded that acids in the range C₁₂-C₁₄ are not effective in restricting silage fermentation and that their effectiveness is not increased by reduction in pH produced by the addition of formic acid. Microbiological assessments largely confirmed these findings.     

Immobilized Glucose Isomerase Use in Mirin Production

The conversion of glucose in mirin (an alcoholic seasoning) to fructose using immobilized glucose isomerase (IGI) was studied in order to produce sweetened mirin without chill haze. The initial conversion velocity (C_m/t_m) with IGI was affected by temperature, pH, and ethanol concentration. This reaction was first order, for which the temperature coefficient (Q₁₀) over the range 20–55°C was 2.0. The value of C_m/t_m was a maximum at pH 8.0, and decreased with an increase in ethanol. Conversions carried out in a continuous column reaction system had C_m/t_m values 36 times higher than that in a batch system. Mirin treated with IGI did not form chill haze.     

Four new SYBR<Superscript>®</Superscript>Green qPCR screening methods for the detection of Roundup Ready<Superscript>®</Superscript>, LibertyLink<Superscript>®</Superscript>, and CryIAb traits in genetically modified products

SYBR^®Green qPCR methods for the detection of the Roundup Ready^® “CP4-EPSPS”, LibertyLink^® “PAT” and “BAR,” and the Bacillus thuringiensis “CryIAb” traits as present in genetically modified organisms (GMO) were developed. Their specificity, sensitivity, and PCR method efficiency were determined. All methods are specific and generate amplicons of 108, 73, 109, and 69 bp, respectively, for “CP4-EPSPS,” “CryIAb,” “PAT,” and “BAR” targets. They perform well at low target levels and can detect down to 5 copies of their respective targets extracted from a sample. The PCR efficiency of the methods ranges between 91 and 109%. Due to their trait-specific nature, these methods allow an efficient screening between the different GMO. In this way, the number of possible GMO candidates present in a sample can be reduced what results in lower global costs due to limiting of further the number of analytical identification steps. The application of these methods in CoSYPS GMO analysis is illustrated using two GEMMA proficiency test samples and a reference material from the GM rapeseed event RF3. This set of SYBR^®Green qPCR trait-specific methods represents a very interesting novel set of GMO analysis methods allowing cost-effective identification of GM materials in products.     

The enzymic breakdown of lipids to volatile and non-volatile carbonyl fragments in disrupted tomato fruits

Physical disruption of tomato fruits results in the degradation of endogenous lipids by hydrolytic and oxidative enzymes. Acyl hydrolase, phospholipase D, lipoxygenase and hydroperoxide cleavage enzymes are active in this tissue. A sequential enzymic pathway is proposed by which tissue lipids are hydrolysed to free (mainly polyunsaturated C₁₈) fatty acids which are subsequently oxidised to their hydroperoxides by lipoxygenase action. The C₁₈ hydroperoxides are cleaved (by an enzyme which is specific for 13-hydroperoxide isomers) to volatile C₆ aldehydes (hexanal or cis-3 hexenal) and non-volatile C₁₂ w-oxoacids. The non-volatile fragment from linoleic acid was identified as 12-oxo-dodec-cis 9-enoic acid.     

LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named “Rapid Amplification of genomic DNA Ends” (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.     

Ohmic heating of peaches in the wide range of frequencies (50 Hz to 1 MHz)

Abstract: The ohmic heating (OH) rate of peaches was studied at fixed electric field strength of 60 V.cm⁻¹, square-shaped instant reversal bipolar pulses, and frequencies varying within 50 Hz to 1 MHz. Thermal damage of tissue was evaluated from electrical admittivity. It showed that the time for half disruption (τ_T) of tissue was required more than 10 h at temperatures below 40 °C. However, cellular thermal disruption occurred almost instantly (τ_T < 1 s) at high temperatures (> 90 °C). Electrical conductivity σ_o and admittivity σ_o* of tissue at T_o= 0 °C and their temperature coefficients (m, m*) were calculated. For freeze–thawed tissues, σ and σ* as well as m and m* were nearly indifferent to the frequency. However, for the intact tissue, both σ_o, σ_o* and m, m* were frequency dependent. For freeze–thawed product, the power factor (P) was approximately equal to 1 and indifferent to the frequency and temperature. On the other hand, strong frequency dependence was observed for intact tissue with the minimum P approximately equal to 0.68 in the range of tens of kHz. The time required to reach a target temperature t_f was evaluated. The t_f increased with frequency up to the middle of the range of tens of kHz and thereafter continuously decreased. Samples exposed to the low-frequency electric field demonstrated faster electro-thermal damage rates. The textural relaxation data supported more intense damage kinetics at low-frequency OH. It has been demonstrated that a combination of high-frequency OH with pasteurization at moderate temperature followed by rapid cooling minimizes texture degradation of peach tissue. Practical Application: In this study, we investigated the electric field frequency effect on the rate of OH of peaches. It was shown that the time required for reaching the target temperature is strongly dependent upon the frequency. Samples exposed to low-frequency OH demonstrated higher electro-thermal damage rates. It has been shown that the combination of high-frequency OH with pasteurization at moderate temperature followed by rapid cooling minimizes texture degradation of peach tissue. Obtained results provide new information on the impact of electric field frequency on OH, which is useful for OH process design.     

Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.