Search

The greatest challenge in the postgenomic era is the description of proteome interactions, such as protein-protein or protein-DNA interactions. Surface plasmon resonance (SPR) is an optical technique in which binding of an analyte to the surface changes the refractive index at the surface/solution interface. Molecular interactions are analysed in real time without a labeling step. Currently, the limit to SPR imaging is the small number of reactions that can be simultaneously analysed. Using a novel grafting technology and a new imaging system, we increased the throughput of SPR imaging. The interaction between p53 and DNA was chosen as a paradigm for validation of this assay. Using a tagged DNA methodology, we simultaneously targeted multiple DNA sequences on a single chip. The interaction between p53 and these DNA sequences was monitored by SPR imaging. Qualitative and quantitative analysis provides results similar to those obtained with conventional technologies.

This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out–of–laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission–sponsored project Good–Food (FP6–IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on–site generation of data that can aid the safe production of high–quality foodstuffs. [ABSTRACT FROM AUTHOR]

Background: In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.Methods: A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).Results: The optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).Conclusions: Although the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples. [ABSTRACT FROM AUTHOR]