Quantitative competitive PCR for the detection of genetically modified soybean and maize

 The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques. Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more than 1% RRS were determined by QC-PCR using various amounts of standard DNA. Received: 13 January 1998 / Revised version: 4 March 1998     

Nested PCR detection of genetically modified soybean in soybean flour, infant formula and soymilk

Due to the Brazilian market introduction of the genetically modified (GM) crop Roundup Ready™ (RR) soybean, the ability to detect GM crops has become a legal necessity. In order to detect the presence of RR soybean, a polymerase chain reaction (PCR) amplification method was evaluated for the detection of RR in soybean mixtures and commercially available soy flour, infant formula and soymilk powder. To detect the presence of RR soybean, a nested PCR resulted in an amplicon of 169 bp, present for all soybean mixed samples containing 0.01-10% GM soybean and absent for 0% GM soybean. None of the analysed infant formulas showed a positive signal after the nested PCR; four out of six soy flour samples and 15 out of 25 soymilk powder samples were positive for the presence of RR soybean. Results show that the nested PCR method used is adequate to determine the presence of GM soybean in the presented products.     

Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNA from two genetically modified organisms

The application of a ligation-dependent probe amplification (LPA) technique to the simultaneous event-specific detection and relative quantification of DNA from genetically modified organisms in foods is described. The system is based on the ligation of synthetic bipartite probes when hybridized to the corresponding target DNA sequence. The ligation products possess lengths characteristic for each target sequence. Universal primer binding sites (PBS) at the 5′ and 3′-ends enable their subsequent competitive amplification using one common pair of primers. The use of one fluorescein (FAM) labeled primer permits amplification products to be separated and detected via capillary electrophoresis. Respective probes were designed to allow the detection of reference genes in the genomes from maize and soya, as well as of event-specific junction regions in the transgenic maize line MON810 and in Roundup Ready soya. Specificity, sensitivity, and the potential of the technique for the relative quantification of recombinant DNA were assayed using mixtures of DNA extracted from certified reference maize and soybean flours. The novel strategy results in a modular system which can be complemented by further probes to broaden the range of target sequences.     

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     

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.     

Transgenes monitoring in an industrial soybean processing chain by DNA-based conventional approaches and biosensors

The development of analytical methods for genetically modified organisms (GMO) screening is of great interest. In particular, since even highly processed GMO-derived food products are covered by new European legislations, a great effort has been devoted to the application of the analytical tests to these products.     

Real-time PCR multiplex method for the quantification of Roundup Ready soybean in raw material and processed food

This paper describes a quantitative real-time multiplex PCR method optimised for the ABI PRISM^® 7700 Sequence Detection System (SDS) and TaqMan^® chemistry for Roundup Ready^® Soybean (RRS) in raw material and processed food. This method has the advantage of performing the amplification of the target taxon lectin gene and the genetically modified (GM) target sequence in the same test tube. The quantification is based on a calibration curve obtained with the DNA extracted from Certified Reference Material standards (CRM IRMM-410: R) in the range 0.1–5% RRS. The method was validated in-house by using CRMs and the applicability was verified on three mixtures of soybean flour at 1, 5, 10% of RRS respectively and on prepared baked products (biscuits and plum cakes). The statistical parameters of the method were found to be satisfactory both for soybean flour and baked products. In the process the absolute LOD and LOQ was 7.1 and 35.5 copy number respectively; the relative LOD and LOQ was 0.03 and 0.01% of RRS respectively.     

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.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

The practical analysis of food: The development of Sakalar quantification table of DNA (SQT-DNA)

Practical and highly sensitive Sakalar quantification table of DNA (SQT-DNA) has been developed for the detection% of species-specific DNA amount in food products. Cycle threshold (Ct) data were obtained from multiple curves of real-time qPCR. The statistical analysis was done to estimate the concentration of standard dilutions. Amplicon concentrations versus each Ct value were assessed by the predictions of targets at known concentrations. SQT-DNA was prepared by using the percentage versus each Ct values. The applicability of SQT-DNA to commercial foods was proved by using sausages containing varying ratios of beef, chicken, and soybean. The results showed that SQT-DNA can be used to directly quantify food DNA by a single PCR without the need to construct a standart curve in parallel with the samples every time the experiment is performed, and also quantification by SQT-DNA is as reliable as standard curve quantification for a wide range of DNA concentrations.