Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene.

Equipe 6International audienceMolecular tools that allow intraspecific quantification and discrimination of pathogen isolates are useful to assess fitness of competitors during mixed infections. However, methods that were developed for quantifying Phytophthora infestans are only specific at the species level. Here, we reported a TaqMan-based real-time PCR assay allowing, according to the specificity of the used probes, an accurate quantification of different proportions of two genetically distinct clones of P. infestans in mixed fractions. Indeed, in addition to a primer specific to P. infestans, two primers and two TaqMan(®) probes that target single-nucleotide polymorphisms located in the Avr3a/avr3a virulence gene sequence were designed. The reliability of the method was tested on serially diluted fractions containing plasmid DNA with either the Avr3a or the avr3a sequences at concentrations ranging from 10(2) to 10(8) copies per μl. Based on its specificity, sensitivity and repeatability, the proposed assay allowed a quantification of the targeted DNA sequence in fractions with a Avr3a/avr3a ratio in the range 1/99 to 99/1. The reliability of the test was also checked for counting zoospores. Applications for future research in P. infestans/host quantitative interactions were also discussed

Comparison of protein quantification and extraction methods suitable for E-coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and sonication-BCA_P combinations

Modification of a commercial dna extraction kit to simultaneously recover rna, safely and rapidly, and to assess molecular biomass of the total and the active part of microbial communities, from soils with diverse mineralogy and carbon content : S11.04-P -15

We have modified a commercial DNA extraction kit for soil to simultaneously co-extract RNA. In this new procedure RNA and DNA are separated by two selective purifications in cascade without the need of DNAase or RNAse digestion. Consequently DNA and RNA are respectively purified from the whole co-extraction solution. Nucleic acids extraction is based on the action of SDS coupled with an efficient bead-beating step, but it does not require any solvent. Avoiding the use of solvents, which are damaging for human health and environmental quality, was one of our most important motivations to develop this protocol. In a second time, we have optimized this protocol to improve the DNA and RNA yield, but kipping those yields below the saturation limit of the kit to assess and quantify the variations of molecular biomass of the total (DNA) and the active (RNA) part of microbial communities in natural samples. We have also introduced a first step of homogenization of soil sample in liquid nitrogen to improve the reliability of the fungal 18S gene sequence quantification. Finally, we have shown that this protocol can be applied to a wide diversity of soils whatever their mineralogy and metal content (2 Ferralsols, 1 Vertisol, 2 Andosols from Madagascar), texture or biomass content (1 poor sandy soil from Congo and one carbon rich temperate soil sample submitted or not to a 1 month cold stress). * E Tournier, L. Amenc and AL. Pablo contributed equally to this study. (Texte intégral

Impact of variance components on reliability of absolute quantification using digital PCR

Background: Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation. Results: We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably. Conclusions: Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments

J Fluorescence

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards

Invalid Forensic Science Testimony and Wrongful Convictions

This is the first study to explore the forensic science testimony by prosecution experts in the trials of innocent persons, all convicted of serious crimes, who were later exonerated by post-conviction DNA testing. Trial transcripts were sought for all 156 exonerees identified as having trial testimony by forensic analysts, of which 137 were located and reviewed. These trials most commonly included testimony concerning serological analysis and microscopic hair comparison, but some included bite mark, shoe print, soil, fiber, and fingerprint comparisons, and several included DNA testing. This study found that in the bulk of these trials of innocent defendants - 82 cases or 60% - forensic analysts called by the prosecution provided invalid testimony at trial - that is, testimony with conclusions misstating empirical data or wholly unsupported by empirical data. This was not the testimony of a mere handful of analysts: this set of trials included invalid testimony by 72 forensic analysts called by the prosecution and employed by 52 laboratories, practices, or hospitals from 25 states. Unfortunately, the adversarial process largely failed to police this invalid testimony. Defense counsel rarely cross-examined analysts concerning invalid testimony and rarely obtained experts of their own. In the few cases in which invalid forensic science was challenged, judges seldom provided relief. This evidence supports efforts to create scientific oversight mechanisms for reviewing forensic testimony and to develop clear scientific standards for written reports and testimony. The scientific community can through an official government entity promulgate standards to ensure the valid presentation of forensic science in criminal cases and thus the integrity and fairness of the criminal process

Invalid Forensic Science Testimony and Wrongful Convictions

This is the first study to explore the forensic science testimony by prosecution experts in the trials of innocent persons, all convicted of serious crimes, who were later exonerated by post-conviction DNA testing. Trial transcripts were sought for all 156 exonerees identified as having trial testimony by forensic analysts, of which 137 were located and reviewed. These trials most commonly included testimony concerning serological analysis and microscopic hair comparison, but some included bite mark, shoe print, soil, fiber, and fingerprint comparisons, and several included DNA testing. This study found that in the bulk of these trials of innocent defendants - 82 cases or 60% - forensic analysts called by the prosecution provided invalid testimony at trial - that is, testimony with conclusions misstating empirical data or wholly unsupported by empirical data. This was not the testimony of a mere handful of analysts: this set of trials included invalid testimony by 72 forensic analysts called by the prosecution and employed by 52 laboratories, practices, or hospitals from 25 states. Unfortunately, the adversarial process largely failed to police this invalid testimony. Defense counsel rarely cross-examined analysts concerning invalid testimony and rarely obtained experts of their own. In the few cases in which invalid forensic science was challenged, judges seldom provided relief. This evidence supports efforts to create scientific oversight mechanisms for reviewing forensic testimony and to develop clear scientific standards for written reports and testimony. The scientific community can through an official government entity promulgate standards to ensure the valid presentation of forensic science in criminal cases and thus the integrity and fairness of the criminal process

A portable device for nucleic acid quantification powered by sunlight, a flame or electricity.

A decentralized approach to diagnostics can decrease the time to treatment of infectious diseases in resource-limited settings. Yet most modern diagnostic tools require stable electricity and are not portable. Here, we describe a portable device for isothermal nucleic-acid quantification that can operate with power from electricity, sunlight or a flame, and that can store heat from intermittent energy sources, for operation when electrical power is not available or reliable. We deployed the device in two Ugandan health clinics, where it successfully operated through multiple power outages, with equivalent performance when powered via sunlight or electricity. A direct comparison between the portable device and commercial qPCR (quantitative polymerase chain reaction) machines for samples from 71 Ugandan patients (29 of which were tested in Uganda) for the presence of Kaposi's sarcoma-associated herpesvirus DNA showed 94% agreement, with the four discordant samples having the lowest concentration of the herpesvirus DNA. The device's flexibility in power supply provides a needed solution for on-field diagnostics