An accurate real-time PCR test for the detection and quantification of cauliflower mosaïc virus (CaMV): applicable in GMO screening

Maher Chaouachi and Marie Noelle Fortabat are contributed equally to this work.     

Modelling the limit of detection in real-time quantitative PCR

The limit of detection (LOD) is a critical performance characteristic of an assay that requires careful evaluation during method validation. However, formal calculations for the LOD do not take into account atypical data sets that are generated from real-time PCR techniques, which can be non-normally distributed, truncated, and heteroscedastic. Experimental data sets for the quantification of genetically modified (GM) material were produced using real-time PCR, in order to model the LOD. A bootstrapping computer simulation calculated the probabilities of detecting PCR positive test results from these data sets, and computer modelling defined a function from the resulting probability plots. The LOD was modelled as a function of sample replication level and cycle threshold values. The broad applicability of this bootstrapping and data modelling approach should be of general interest to laboratories conducting trace-level detection.     

A simulation approach to assess the minimal number of real-time PCR replicates for GM quantification

The quantification of genetically modified (GM) ingredients in food and feed typically uses real-time quantitative PCR (RT-QPCR). In recent years a multitude of new RT-QPCR assays have facilitated increased method performance. The level of sample replication within these assays is a fundamental aspect that needs to be considered to produce results with high confidence. In this paper we describe the use of a modelling approach as applied to GM and RT-QPCR data sets, to objectively assesses the effect of different levels of PCR replication in terms of the variability associated with a result, and demonstrate that it is possible to use a reduced level of replication without a subsequent reduction in the confidence of a result. Using an example data set, we show it is possible to reduce the sample level of replication from six to three PCR replicates, without a significant change in the mean value of the result. The use of such an approach can facilitate the use of the minimum number of replicates in order to produce an accurate result, thus saving on important resources involved in quantification assays.     

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.     

A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food

A real-time polymerase chain reaction (PCR)-based method for the detection of the walnut (Juglans regia) component in food is described here. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with walnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the jug r2, a major allergen gene of walnut. The method was positive for 8 varieties of walnut and negative for all other tested plant materials used in food industry, including pecan nuts. The intrinsic detection limit of the method was 0.24 ng walnut DNA. Using a series of model pastry samples with defined walnut contents, a practical detection limit of 0.01% walnut content was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples (bakery and confectionery products), out of which two cakes were found to contain walnuts although they were not adequately labelled. The presented PCR method is useful for sensitive and selective detection of walnuts in food samples and can be performed in one working day.     

基于竞争性等位基因特异性PCR技术的麦芽品种纯度定性和定量检测

利用测序技术筛选和确认4?个可以区分7?种大麦麦芽的单核苷酸多态性（single nucleotide polymorphisms,SNP）位点,并建立区分图谱。同时,利用竞争性等位基因特异性聚合酶链式反应检测技术对预混样品进行定量测试,其检测的结果与真实性之间相对误差小于3%。该技术的建立对大麦麦芽纯度检测提供方法,对大麦麦芽SNP指纹数据库的完善提供了数据支持。     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

Comparing different gas chromatographic methods for the quantification of bisphenol A (BPA) trace levels in paper and cardboard products from the market

Bisphenol A (BPA; 4,4?-(propane-2,2-diyl)diphenol), a suspected endocrine disruptor with weak estrogenic activity, is used in a variety of consumer products, including paper and cardboard products used as food contact materials. The present study compared four different gas chromatographic methods for the analysis of BPA in paper and cardboard food packages. Eighteen different food packages were extracted and BPA was determined using two different derivatisation reactions – trimethylsilylation with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and halide alkylation with pentafluorobenzoyl chloride (PFBOCl) – and four different separation and detection techniques. The BSTFA derivatives were quantified with (1) GC-MS in single-ion monitoring (SIM) mode with electron ionisation (EI-GC-MS) and (2) GC-MS/MS in multiple reaction monitoring (MRM) mode using electron ionisation (EI-GC-MS/MS); while the PFBOCl derivatives were quantified with (3) GC-MS using electron ionisation (EI-GC-MS) as well as (4) GC-MS with negative chemical ionisation (NCI-GC-MS). All developed methods showed good linearity (R² > 0.9938), precision (CV < 4.5% for reproducibility; CV < 2.2% for repeatability) and sensitivity, with limits of detection (LODs) between 0.02 µg kg^?1 for the pentafluorobenzoyl derivatives measured with the NCI-GC-MS method and 6 µg kg^?1 for the pentafluorobenzoyl derivatives determined with EI-GC-MS. Levels of BPA in the samples were in agreement for all methods, ranging from values below the limit of quantitation (LOQ) to 11.9 mg kg^?1 paper. In a last step, the maximum potential migration into food products was calculated for all tested paper and cardboard samples, assuming a ‘worst case’ scenario of 100% migration.     

Investigation of test performance of the dual reminder A-Not A (DR A-Not A) in comparison to 3-AFC for discriminating samples of drinking water

Although the relative performance of sensory discrimination methods is well theorized in Thurstonian modeling and signal detection theory (SDT), empirical research is needed to investigate how such theorized models could be validated in complex sensory testing situations of food and beverages. This paper presents a practical procedure to utilize the existing SDT-based A-Not A sensory discrimination model based on a beta-strategy for detecting the off-sensory quality of samples of drinking water and validates the model for effective and efficient sensory quality management of food and beverages. Dual reminder A-Not A (DR A-Not A) using two tastings of the reference stimulus before evaluating every test stimulus is proposed as the optimal test procedure because it uses more sensitive test sequences and facilitates subjects’ familiarization with the standard quality of the reference. To test the performance of DR A-Not A, 3-AFC, which also uses three stimuli and is the recommended method for detecting odor or taste stimuli by ASTM, was used as a control method. In Experiment 1, 98 subjects each performed both DR A-Not A and 3-AFC. Based on these results, only sensitive subjects were selected for the next experiment, in which they were divided into two equally well performing groups. In Experiment 2, each group performed either DR A-Not A or 3-AFC over three repeated sessions. The results confirmed that the A-Not A beta-strategy was adopted for DR A-Not A and that its test performance was improved over replications. These results suggest that although DR A-Not A is an unspecified difference test method and does not use a forced-choice task, embedding the familiarization procedure for the reference renders it an effective sensory difference method for the sensory quality management of drinking water.