Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Recent advances in capillary electrophoresis methods for food analysis

This review article addresses recent advances in the analysis of foods and food components by capillary electrophoresis (CE). CE has found application to a number of important areas of food analysis, including quantitative chemical analysis of food additives, biochemical analysis of protein composition, and others. The speed, resolution and simplicity of CE, combined with low operating costs, make the technique an attractive option for the development of improved methods of food analysis for the new millennium.

The best of both worlds: Producing national statistics using participatory methods

This paper presents our experience with combining statistical principles and participatory methods to generate national statistics. The methodology was developed in Malawi during 1999–2002. We demonstrate that if PRA is combined with statistical principles (including probability-based sampling and standardization), it can produce total population statistics and estimates of the proportion of households with certain characteristics (e.g., poverty). It can also provide quantitative data on complex issues of national importance such as poverty targeting. This approach is distinct from previous PRA-based approaches, which generate numbers at community level but only provide qualitative information at national level.

Quantitative and qualitative differences in morphological traits revealed between diploid Fragaria species

Background and Aims: The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. Methods: A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. Key Results: Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. Conclusions: Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group.

Quantitative comparison between the use of 3D vs 2D visualization tools to present building design proposals to non-spatial skilled end users

Information technology in construction (ITC) has been gaining wide acceptance and is being implemented in the construction research domains as a tool to assist decision makers. Most of the research into visualization technologies (VT) has been on the wide range of 3D and simulation applications suitable for construction processes. Despite its development with interoperability and standardization of products, VT usage has remained very low when it comes to communicating and addressing the needs of building end-users (BEU). This paper argues that building end users are a source of experience and expertise that can be brought into the briefing stage for the evaluation of design proposals. It also suggests that the end user is a source of new ideas promoting innovation. In this research a positivistic methodology that includes the comparison of 3D models and the traditional 2D methods is proposed. It will help to identify "how much", if anything, a non-spatial specialist can gain in terms Of "understanding" of a particular design proposal presented, using both methods.

Contextualist research: Iterating between methods while following an empirically grounded approach

An alternative approach to research is described that has been developed through a succession of significant construction management research projects. The approach follows the principles of iterative grounded theory, whereby researchers iterate between alternative theoretical frameworks and emergent empirical data. Of particular importance is an orientation toward mixing methods, thereby overcoming the existing tendency to dichotomize quantitative and qualitative approaches. The approach is positioned against the existing contested literature on grounded theory, and the possibility of engaging with empirical data in a “theory free” manner is discounted. Emphasis instead is given to the way in which researchers must be theoretically sensitive as a result of being steeped in relevant literatures. Knowledge of existing literatures therefore shapes the initial research design; but emergent empirical findings cause fresh theoretical perspectives to be mobilized. The advocated approach is further aligned with notions of knowledge coproduction and the underlying principles of contextualist research. It is this unique combination of ideas which characterizes the paper's contribution to the research methodology literature within the field of construction management. Examples are provided and consideration is given to the extent to which the emergent findings are generalizable beyond the specific context from which they are derived.

Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides

Aims: To develop a quantitative equation [prebiotic index ( PI)] to aid the analysis of prebiotic fermentation of commercially available and novel prebiotic carbohydrates in vitro, using previously published fermentation data. Methods: The PI equation is based on the changes in key bacterial groups during fermentation. The bacterial groups incorporated into this PI equation were bifidobacteria, lactobacilli, clostridia and bacteroides. The changes in these bacterial groups from previous studies were entered into the PI equation in order to determine a quantitative PI score. PI scores were than compared with the qualitative conclusions made in these publications. In general the PI scores agreed with the qualitative conclusions drawn and provided a quantitative measure. Conclusions: The PI allows the magnitude of prebiotic effects to be quantified rather than evaluations being solely qualitative. Significance and Impact of the Study: The PI equation may be of great use in quantifying prebiotic effects in vitro. It is expected that this will facilitate more rational food product development and the development of more potent prebiotics with activity at lower doses.

Quantitative analysis of accuracy of an inertial/acoustic 6DOF tracking system in motion

An increasing number of neuroscience experiments are using virtual reality to provide a more immersive and less artificial experimental environment. This is particularly useful to navigation and three-dimensional scene perception experiments. Such experiments require accurate real-time tracking of the observer's head in order to render the virtual scene. Here, we present data on the accuracy of a commonly used six degrees of freedom tracker (Intersense IS900) when it is moved in ways typical of virtual reality applications. We compared the reported location of the tracker with its location computed by an optical tracking method. When the tracker was stationary, the root mean square error in spatial accuracy was 0.64 mm. However, we found that errors increased over ten-fold (up to 17 mm) when the tracker moved at speeds common in virtual reality applications. We demonstrate that the errors we report here are predominantly due to inaccuracies of the IS900 system rather than the optical tracking against which it was compared. (c) 2006 Elsevier B.V. All rights reserved.

Methods for comparing and constraining models of dendritic spines

Physiological evidence using Infrared Video Microscopy during the uncaging of glutamate has proven the existence of excitable calcium ion channels in spine heads, highlighting the need for reliable models of spines. In this study we compare the three main methods of simulating excitable spines: Baer & Rinzel's Continuum (B&R) model, Coombes' Spike-Diffuse-Spike (SDS) model and paired cable and ion channel equations (Cable model). Tests are done to determine how well the models approximate each other in terms of speed and heights of travelling waves. Significant quantitative differences are found between the models: travelling waves in the SDS model in particular are found to travel at much lower speeds and sometimes much higher voltages than in the Cable or B&R models. Meanwhile qualitative differences are found between the B&R and SDS models over realistic parameter ranges. The cause of these differences is investigated and potential solutions proposed.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Extracting quantitative structural parameters for disordered polymers from neutron scattering data

The organization of non-crystalline polymeric materials at a local level, namely on a spatial scale between a few and 100 a, is still unclear in many respects. The determination of the local structure in terms of the configuration and conformation of the polymer chain and of the packing characteristics of the chain in the bulk material represents a challenging problem. Data from wide-angle diffraction experiments are very difficult to interpret due to the very large amount of information that they carry, that is the large number of correlations present in the diffraction patterns.We describe new approaches that permit a detailed analysis of the complex neutron diffraction patterns characterizing polymer melts and glasses. The coupling of different computer modelling strategies with neutron scattering data over a wide Q range allows the extraction of detailed quantitative information on the structural arrangements of the materials of interest. Proceeding from modelling routes as diverse as force field calculations, single-chain modelling and reverse Monte Carlo, we show the successes and pitfalls of each approach in describing model systems, which illustrate the need to attack the data analysis problem simultaneously from several fronts.

A flow system for the on-line quantitative measurement of the retention of dosage forms on biological surfaces using spectroscopy and image analysis

Measuring the retention, or residence time, of dosage forms to biological tissue is commonly a qualitative measurement, where no real values to describe the retention can be recorded. The result of this is an assessment that is dependent upon a user's interpretation of visual observation. This research paper outlines the development of a methodology to quantitatively measure, both by image analysis and by spectrophotometric techniques, the retention of material to biological tissues, using the retention of polymer solutions to ocular tissue as an example. Both methods have been shown to be repeatable, with the spectrophotometric measurement generating data reliably and quickly for further analysis.

Quantitative precipitation estimation over ocean using Bayesian approach from microwave observations during the typhoon season

We have developed a new Bayesian approach to retrieve oceanic rain rate from the Tropical Rainfall Measuring Mission (TRMM) Microwave Imager (TMI), with an emphasis on typhoon cases in the West Pacific. Retrieved rain rates are validated with measurements of rain gauges located on Japanese islands. To demonstrate improvement, retrievals are also compared with those from the TRMM/Precipitation Radar (PR), the Goddard Profiling Algorithm (GPROF), and a multi-channel linear regression statistical method (MLRS). We have found that qualitatively, all methods retrieved similar horizontal distributions in terms of locations of eyes and rain bands of typhoons. Quantitatively, our new Bayesian retrievals have the best linearity and the smallest root mean square (RMS) error against rain gauge data for 16 typhoon overpasses in 2004. The correlation coefficient and RMS of our retrievals are 0.95 and ~2 mm hr-1, respectively. In particular, at heavy rain rates, our Bayesian retrievals outperform those retrieved from GPROF and MLRS. Overall, the new Bayesian approach accurately retrieves surface rain rate for typhoon cases. Accurate rain rate estimates from this method can be assimilated in models to improve forecast and prevent potential damages in Taiwan during typhoon seasons.