Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.     

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.     

A DANREF certified reference plastic for measurement of overall migration into the food simulant olive oil by single sided testing

A reference material for the determination of overall migration from a plastic coextrudate into the fatty food simulant olive oil was produced and certified in an interlaboratory study. The analyses were carried out according to the ENV 1186 standard from the European Committee for Standardization (CEN) [ 1, 2, 3] with exposure of the coextrudate to olive oil for 10 days at 40 degrees C. After an initial preliminary interlaboratory study eight laboratories participated in the certification round, and two different methods were used to obtain single sided exposure of the plastic to the oil. The certified value was determined as the mean of laboratory mean values. No outliers were found. A reference value of 8.6 mg/dm2 +/- 1.4 mg/dm2 (+/- half width of the 95% confidence interval) was obtained which is within the range relevant for the regulatory limit (10 mg/ dm2), making this reference material suitable for laboratories measuring according to the EU overall migration limit [4]. The material has been found stable over 45 months.     

A DANREF certified reference plastic for measurement of overall migration into the food simulant olive oil by single sided testing

A reference material for the determination of overall migration from a plastic coextrudate into the fatty food simulant olive oil was produced and certified in an interlaboratory study. The analyses were carried out according to the ENV 1186 standard from the European Committee for Standardization (CEN) [1, 2, 3] with exposure of the coextrudate to olive oil for 10 days at 40?°C. After an initial preliminary interlaboratory study eight laboratories participated in the certification round, and two different methods were used to obtain single sided exposure of the plastic to the oil. The certified value was determined as the mean of laboratory mean values. No outliers were found. A reference value of 8.6 mg/dm²± 1.4 mg/dm² (± half width of the 95% confidence interval) was obtained which is within the range relevant for the regulatory limit (10 mg/ dm²), making this reference material suitable for laboratories measuring according to the EU overall migration limit [4]. The material has been found stable over 45 months.     

Qualitative PCR method for Roundup Ready soybean: interlaboratory study

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.     

Proficiency testing as a tool for implementing internal quality control: the case of ochratoxin A in cocoa powder

This paper describes the interlaboratory study aimed at assessing the performance of 18 laboratories (14 national and 4 European) for Ochratoxin A (OTA) determination in cocoa powder samples. The study was tested at three levels of OTA covering the range in which presumably European regulatory limits could fall in the near future. For the extraction step, almost all laboratories used an aqueous solution of sodium hydrogen carbonate with the exception of one laboratory using dichloromethane consistently with the ELISA procedure adopted in the study. The clean-up step was performed by utilizing the immunoaffinity columns by the two main manufacturers (R-Biopharm Rhone and VICAM) and for the quantitative analysis, HPLC was used by all the participants except one using ELISA. From the output of the study, it can be concluded that at low level (0.19 μg/kg) 10 out of 18 (56%), at medium level (0.45 μg/kg) 11 out of 18 (61%), and at high level (1.45 μg/kg) 12 out of 18 (67%) results fell within the satisfactory ranges. This interlaboratory study provides an estimate of the performance of national and European laboratories involved in OTA determination in cocoa powder samples, which sounds extremely valuable in view of potential future legislation by the European Commission.     

EAL interlaboratory comparison Wa1 – Testing of potable water

 The accreditation of laboratories has emphasized the use of interlaboratory comparisons as a tool to monitor the comparability and accuracy of results laboratories produce. An interlaboratory comparison for water laboratories was organized among European Cooperation for Accreditation (EA) member countries; 30 laboratories, 7 of which were not accredited, from 14 European countries participated in this intercomparison. All the laboratories were chosen by the appropriate national accreditation bodies, with the instruction to select as participants those laboratories which act as national reference laboratories in this field. About 90% of the data collected was considered satisfactory after statistical treatment. Non-accredited laboratories performed as well as accredited laboratories. The laboratories were asked to take corrective action and report the corrections to the accreditation bodies. A great variation in the reported uncertainties of the results was observed. There seems to be a need to organize EA interlaboratory comparisons for national reference laboratories analysing water. It is obvious that even reference laboratories need training in how to estimate the uncertainty of results. Received: 22 July 1998 · Accepted: 21 September 1998     

The influence of different evaluation techniques on the results of interlaboratory comparisons

The influence of different evaluation techniques on the results of an interlaboratory comparison for the determination of nutrients in ground- and surface water was investigated. The outlier-test procedure was found to influence the interlaboratory standard deviations (SDs), but not the averages. It was shown that even small differences in the numbers of outliers detected can change the SD severely. Comparing the outlier-test procedures of Hampel, Grubbs and Graf-Henning, it was found that Hampel's test detected the most outliers, thus generally resulting in smaller SDs between interlaboratory comparisons. The Graf-Henning test detected the fewest outliers and its application resulted in the highest SDs of the three test procedures investigated. The comparison of different summarising indices, namely the rescaled sum of z-scores, average of absolute z-scores and average deviation showed no comparability. Possibilities to improve the comparability of interlaboratory comparisons and to minimise misunderstandings are suggested.     

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.     

Determination of the carbon-13 content of sugars and pulp from fruit juices by isotope-ratio mass spectrometry (internal reference method). A European interlaboratory comparison

An interlaboratory comparison by ¹³C-isotope ratio mass spectrometry was organised by working group No. 1 of the European Commission of Standardisation (CEN/TC 174) in order to define the repeatability (r) and the reproducibility (R) of the ¹³C determination in sugars and pulp, isolated from the same fruit juice. Six unlabelled juices were analysed in 19 laboratories in the European Union, Australia and the USA. Three samples were authentic juices (orange, grapefruit, pineapple) and the remaining samples were the same juices with an addition of sugar at 15 g l⁻¹ (orange, pineapple) or 11.8 g l⁻¹ (grapefruit). The sugar was prepared by mixing equal amounts of beet and cane sucroses. The different laboratories used the same experimental protocol under different conditions (operator, conversion system for CO₂ preparation, mass spectrometer). The results for the sugars (r = 0.27%., R = 0.82%.) were comparable to those from an earlier ring test (r = 0.30%., R = 0.70%.), while the results for the pulp presented higher interlaboratory variability (r = 0.38%., R = 1.89%.) possibly due to experimental difficulties. Nevertheless, the RSD_R for free sugars ranged 0.9–3.2% and for pulp, 0.9–9.3%. On the basis of the Horwitz equation and taking account of the concentration of the isotopes measured, an RSD_R of 7–8% might have been expected. The experimentally determined RSD_R values were less than twice the theoretically expected values, which is a criterion of an acceptable method. The results were therefore considered to be acceptable by specialists in the field of isotopic analysis, and the method applied was found to improve the sensitivity of the ¹³C determination for the detection of sugar addition in fruit juices.     

News from CEN/BT/WG 122 uncertainty and EA expert group on uncertainty

Since the publication in 2000 of the standard ISO/IEC 17025 "General requirements for the competence of testing and calibration laboratories", the concept of uncertainty of measurement or test results seems to be a key issue for laboratories and their clients. Two working groups are trying to implement this concept in testing activities. The CEN/BT/WG122 has proposed to the technical bureau of CEN a series of recommendations on how to introduce this concept in European standards and the EA expert group on uncertainty has prepared a guidance document in order to help the testing community in uncertainty evaluation. The objective of this paper is to report the current situation of these two groups.     

Interlaboratory comparison study for the determination of methyl tert-butyl ether in water

This is the first publication which describes the evaluation of the analytical performance and state-of-the-art of the determination of methyl tert-butyl ether (MTBE) in water at ng L^–1 concentrations. An interlaboratory comparison study for the determination of MTBE in water was carried out. Twenty-eight laboratories from seven European countries participated in the study. Twenty of those finally transmitted results to the organiser. Italian spring water, containing no detectable amounts of MTBE was fortified to yield two samples with MTBE concentrations of 0.074±0.004 µg L^–1 and 0.256±0.010 µg L^–1. The laboratories applied their regular in-house methods to analyse the water samples. Static headspace, Purge & Trap, solid-phase microextraction (SPME) or direct aqueous injection were used as sample preparation techniques. Subsequent separation and detection of MTBE were performed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/flame ionisation detection (GC/FID). After rejection of outliers, the overall arithmetic mean of laboratory results corresponded to recoveries of 78±20% (Sample A) and 88±20% (Sample B) of the reference concentrations. The between laboratory coefficients of variation (CV) were 32% and 31%, respectively. The organisation of the study and quality assurance measures at the organiser's laboratory are described. Moreover, the measurement results of the participants and the analytical methods used for the determination of MTBE are presented and the correlation between selected method parameters and data quality is discussed.     

Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial

The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard.     

国际实验室间转基因产品检测能力验证的研究

以转基因大豆为例，介绍开展APLAC T034国际间实验室转基因检测能力验证的项目。对于样品量值(添加值)的设定进行试验，对样品的均匀性和稳定性进行检验，并对参试实验室的测试结果进行系统的评价和分析。影响转基因产品测试结果的关键因素是检测方法、基因组DNA提取的质量、PCR反应体系中模板DNA的加入量．以及是否使用标准物质作为质控对照和使用定量标准物质求出相关系数对定量检测结果进行校正。     

Interlaboratory validation of the Vidas SET2 kit for detection of staphylococcal enterotoxins in milk products

An earlier intralaboratory validation study based on the EN ISO 16140 Standard conducted by the Community Reference Laboratory for coagulase-positive staphylococci including Staphylocococcus aureus showed that, after an extraction step using dialysis concentration, the Vidas SET2 detection kit could be used to screen staphylococcal enterotoxins in milk and milk products. In order to fully validate Vidas SET2, an interlaboratory study was organized. Six freeze-dried samples and 3 ready-to-use concentrated extracts were analyzed by 21 laboratories according to the method, including a detection with Vidas SET2. Results did not show false-positive or -negative results. Accordance and concordance parameters were equal to 100%, corresponding to a concordance odds ratio of 1. This interlaboratory study confirmed the satisfactory outcome of the preliminary tests and of the intralaboratory study performed previously. The Vidas SET2 detection kit can be used as a method for the detection of staphylococcal enterotoxins in milk and milk products as well as the Transia Plate SET detection kit in the European screening method for official control purposes, after an extraction step followed by dialysis concentration.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Comparison of different statistical methods for evaluation of proficiency test data

In analytical chemistry, proficiency testing usually consists in tests that laboratories conduct under routine conditions and report the result to the PT provider who then converts the result to a score which helps the participant to assess the accuracy of the result. The aim of this work is to show PT providers, accreditations bodies, and participating laboratories that different scoring results can be achieved depending on the evaluation system selected. The influence of different evaluation techniques on the results of an interlaboratory comparison for determination of gold in precious metals alloys was investigated. Results from 19 participating laboratories were evaluated by means of the three procedures: (1) classical statistical approach—outliers detection; (2) robust methods—(2A) robust procedure and (2B) ISO 13528; and (3) fitness for purpose. Evaluation of the same PT data revealed very interesting issues depending on the different scoring systems that were used and the robustness of the statistical methods used for detecting outliers. As a general rule, laboratories with scoring Z > 2 offered clearly poorer performance in robust approaches than classical ones. In order to support this first evidence, we evaluated a second data set with results from 24 laboratories (mercury from soil samples) by means of the four mentioned approaches. Selection and comparison of different scoring systems must be done very carefully, because sometimes they are not the best approach for studying the data population or the more appropriate one for evaluating the distribution of the data. Finally it should be taken into account that sometimes the robust scoring systems are not always suitable for evaluating the results of some PT schemes.     

Method validation for determination of the 15 European-priority polycyclic aromatic hydrocarbons in primary smoke condensates by gas chromatography/mass spectrometry: interlaboratory study

A collaborative study was conducted to validate an analytical method for quantification of the 15 polycyclic aromatic hydrocarbons (PAHs) regarded in 2002 as a health concern by the former Scientific Committee on Food of the European Commission (SCF) in primary smoke condensates. The method is based on gas chromatography/mass spectrometry of a cyclohexane extract with solid-phase cleanup through silica gel. The analytes were detected in the selected-ion monitoring mode and quantified by using 3 isotopically labeled internal standard compounds. Seventeen laboratories participated in the collaborative validation study, of which 12 reported valid results. The data were subjected to Cochran, single Grubbs, and double Grubbs tests for statistical outliers. A maximum of 2 outliers was eliminated before further statistical evaluation of the method performance characteristics. Depending on the analyte, the results showed relative standard deviations for repeatability between 4.2 and 30% and for reproducibility from 9.9 to 60%. The recoveries varied between about 50 and 85%, except those for cyclopenta[cd]pyrene, dibenzo[a,l]pyrene, and dibenzo[a,h]pyrene. Nevertheless, because Commission Directive 2005/10/EC allows for a recovery range of 50-120% for (BaP) benzo[a]pyrene in various foods, it can be concluded that the method performs appropriately within the analytical range between 5 and 25 microg/kg of primary smoke condensate. For BaP the validated analytical range covered 5-20 microg/kg, and for benzo[a]anthracene (BaA) 10-25 microg/kg. The method is suitable for monitoring BaP and BaA at their respective maximum permitted levels of 10 and 20 microg/kg. Three analytes, benzo[b]-, benzo[j]-, and benzo[k]-fluoranthene could not be separated by all of the participants and were therefore treated as the sum. Nevertheless, with this method the pattern of the respective concentrations of these 15 PAHs can be monitored in primary smoke condensate as suggested by the SCF.     

Increased efficacy for in-house validation of real-time PCR GMO detection methods

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD_r and RSD_R) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.     

Results of a European interlaboratory study for the determination of oxytetracycline in pig muscle by HPLC.

An interlaboratory study was organised in 1996 in order to determine laboratory testing performance for measuring oxytetracycline (OTC) and 4-epioxytetracycline (4-epiOTC) residues in pig muscle. The organisation was designed according to the 'International Harmonised Protocol for Proficiency Testing of (Chemical) Analytical Laboratories' produced by the Association of Official Analytical Chemists (AOAC International) and the International Union of Pure and Applied Chemistry (IUPAC). Fourteen National Reference Laboratories (NRLs) out of the 15 asked to participate agreed to analyse the samples. These laboratories were from 13 different European Union (EU) Member States and each participant was allowed to use the extraction method of their own choice but had to use liquid chromatography as the analytical technique. Most of the methods used were based on UV detection (simple wavelength or diode-array detection) but some involved fluorimetric detection. The production of incurred samples was prepared on-site. The OTC and 4-epiOTC concentrations present in the samples were determined by taking the mean of the results (excluding outliers) and the deviation of each result from the assigned value was measured. The performance of the laboratories was evaluated by calculating the 'z-scores'. The results were globally satisfactory and showed that all 14 laboratories were capable of determining OTC and 4-epiOTC in pig muscle with satisfactory accuracy. Only two laboratories obtained a questionable result in terms of repeatability.