Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.)

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.     

Development of real-time polymerase chain reaction assay with TaqMan probe for the quantitative detection of infectious hypodermal and hematopoietic necrosis virus from shrimp

An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.     

The detection of T-Nos,a genetic element present in GMOs,by cross-priming isothermal amplification with real-time fluorescence

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10³ copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations

The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.     

Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5 × 10⁵ to 1.5 × 10¹⁰ copies). Linear relationship of the standard curve (R² = 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.     

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL^?1 milk and 0.4 ng mL^?1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n?=?8) or non-transgenic (n?=?7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25 % in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias ?13.27 to 13.05 %). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO. Graphical Abstract

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Simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes using oscillatory-flow multiplex PCR

An oscillatory-flow multiplex PCR method in a capillary microfluidic channel has been developed for the simultaneous determination of pre-purified DNA of multiple foodborne bacterial pathogens. The PCR solution passes three temperature zones in an oscillatory manner. The thermal stability and sample evaporation of the microfluidic device were investigated. Under controlled conditions, a highly efficient multiplex PCR was accomplished as demonstrated for the simultaneous amplifications of 278 bp, 168 bp, and 106 bp DNA fragments within 35 min after 35 cycles for simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. This is much shorter than that of a conventional PCR machine. The detection limits of bacterial genome DNA for the three species are about 399, 314, and 626 copies per μL, respectively. This is comparable to those obtained with the conventional multiplex PCR. Consequently, the oscillatory-flow multiplex PCR technology holds good potential for rapid amplification and detection of nucleic acids of microbial foodborne pathogens.

A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15 nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody–DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5 pg L^?1 to 10 ng L^?1, and the limit of detection (LOD) was 1.72 pg L^?1. Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82 % to 112 %, and the coefficient of variation (CV) was acceptable. The results were compared with GC–ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

Detection of the food allergen celery via loop-mediated isothermal amplification technique

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0 % for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.     

Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD_abs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

Disposable sensors for rapid screening of mutated genes

A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH₃)₅?L]²⁺, where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at –0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/μL PCR product can be detected.     

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.     

Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms

Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.     

Influence of Different Processing Treatments on the Detectability of Nucleic Acid and Protein Targets in Transgenic Soybean Meal

Influences of dry heating, wet heating, and extrusion on the degradation of DNA and protein from transgenic soybean meal (TSM) were analyzed using qualitative PCR, quantitative real-time PCR (qPCR), indirect enzyme-linked immunosorbent assay (iELISA), and Western blot. The 414-bp Lectin gene was thoroughly degraded after dry heating between 75 and 90 °C for 30 min, wet heating, and extrusion at 165 °C with 39 % moisture content. The 483-bp CP4-EPSPS gene was not detected after dry heating, wet heating, and extrusion at 120 °C with 39 % moisture content. The degradation ratios of both Lectin and CP4-EPSPS genes increased from 0.4 to 92.1 % and 6.1 to 84.0 % as temperatures rose from 90 to 165 °C. iELISA results of the extruded TSM showed that the CP4-EPSPS protein content was reduced from 4.19 to 0.54 % as temperatures rose from 90 to 150 °C and was not detectable at 165 °C. Western blot results also showed the degradation of CP4-CPSPS protein after extrusion. Our results showed that temperature played an essential role in DNA and protein degradation, and the content of genetically modified organism (GMO) products may be changed after processing and could not reflect the initial content of the products.     

Duplex probes: a new approach for the detection of specific nucleic acids in homogenous assays

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based on the use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resulting fluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize the presence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combined with real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initial target molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.     

A new genosensor for meningococcal meningitis diagnosis using biological samples

In this work, a new electrochemical biosensor for DNA detection of bacterial meningitis is proposed. The system is based on specific DNA fragments from the Neisseria meningitidis genome as a probe incorporated on graphite electrodes modified with poly(4-aminophenol). Detection of a complementary oligonucleotide sequence, a specific 710-base pair amplicon, and the genomic DNA of bacteria was carried out by differential pulse voltammetry, using ethidium bromide as an electroactive indicator of hybridization. The complementary oligonucleotide and the genomic DNA of Neisseria meningitidis were quantified by the genosensor, showing detection limits of 0.6 ng μL^?1 and about 6 ng μL^?1, respectively. Morphological differences were observed between hybridized and unhybridized surfaces by atomic force microscopy. The biosensor showed high selectivity, discriminating non-specific targets, and high stability retaining over 98% of its original activity after 120 days of storage. The bioelectrode was effective in discriminating the genomic DNA in samples with human serum without significant interference, proving to be an interesting platform for meningococcal meningitis diagnosis.     

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10⁶ copies.     

Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both delta C_T and standard curve approaches are tested. Delta C_T methods are based on direct comparison of measured C_T values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C_T method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.