Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools

AbstractThe detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3–9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a ‘hyper sensitive ELISA’ for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant

Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus-, species- and strain-specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI-TOF analysis. Copyright (C) 2004 John Wiley Sons, Ltd

Immuno-quantitative polymerase chain reaction for detection and quantitation of prion protein.

&lt;p&gt;Immuno-polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno-quantitative PCR (iqPCR), exploiting real-time PCR technology, in order to improve this immuno-detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post-mortem stage.&lt;/p&gt;</p