A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BACKGROUND: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 10(8 )copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens

Trapping in irradiated p-on-n silicon sensors at fluences anticipated at the HL-LHC outer tracker

The degradation of signal in silicon sensors is studied under conditions expected at the CERN High-Luminosity LHC. 200 $\mu$m thick n-type silicon sensors are irradiated with protons of different energies to fluences of up to $3 \cdot 10^{15}$ neq/cm$^2$. Pulsed red laser light with a wavelength of 672 nm is used to generate electron-hole pairs in the sensors. The induced signals are used to determine the charge collection efficiencies separately for electrons and holes drifting through the sensor. The effective trapping rates are extracted by comparing the results to simulation. The electric field is simulated using Synopsys device simulation assuming two effective defects. The generation and drift of charge carriers are simulated in an independent simulation based on PixelAV. The effective trapping rates are determined from the measured charge collection efficiencies and the simulated and measured time-resolved current pulses are compared. The effective trapping rates determined for both electrons and holes are about 50% smaller than those obtained using standard extrapolations of studies at low fluences and suggests an improved tracker performance over initial expectations

Mechanical stability of the CMS strip tracker measured with a laser alignment system

Peer reviewe

Characterisation of irradiated thin silicon sensors for the CMS phase II pixel upgrade

The high luminosity upgrade of the Large Hadron Collider, foreseen for 2026, necessitates the replacement of the CMS experiment's silicon tracker. The innermost layer of the new pixel detector will be exposed to severe radiation, corresponding to a 1 MeV neutron equivalent fluence of up to Phi(eq) = 2x10(16) cm(-2), and an ionising dose of approximate to 5 MGy after an integrated luminosity of 3000 fb(-1). Thin, planar silicon sensors are good candidates for this application, since the degradation of the signal produced by traversing particles is less severe than for thicker devices. In this paper, the results obtained from the characterisation of 100 and 200 mu m thick p-bulk pad diodes and strip sensors irradiated up to fluences of Phi(eq) = 1.3 x 10(16) cm(-2) are shown.Peer reviewe

P-Type Silicon Strip Sensors for the new CMS Tracker at HL-L-HC

Abstract: The upgrade of the LHC to the High-Luminosity LHC (HL-LHC) is expected to increase the LHC design luminosity by an order of magnitude. This will require silicon tracking detectors with a significantly higher radiation hardness. The CMS Tracker Collaboration has conducted an irradiation and measurement campaign to identify suitable silicon sensor materials and strip designs for the future outer tracker at the CMS experiment. Based on these results, the collaboration has chosen to use n-in-p type silicon sensors and focus further investigations on the optimization of that sensor type

Spiral and square microstructured surfaces: the effect of the decreasing size of photo-immobilised hyaluronan domains on cell growth

Abstract Spiral and squared micropatterned surfaces of decreasing dimensions were realized by photo-immobilizing a photoreactive hyaluronan (Hyal) derivative on silanized glass substrates. The microstructured surfaces were observed by atomic force microscope and scanning electron microscope. Scanning electron microscope analysis revealed the presence of a spiral (ranging from 100 microm down to 1 microm in the central part) and a square pattern consisting of a central square of 100 microm x 100 microm and squares of different dimensions decreasing from the centre to the edges of the micropatterned area (2 microm x 1 microm). Three cell types were tested on all the microstructured surfaces: human coronary artery endothelial cells (HCAEC), human dermal fibroblasts (C54), and NIH 3T3 fibroblasts. Cell adhesion analysis demonstrated that HCAEC and C54 did not adhere to the immobilized Hyal on silanized glass but adapted their shape to the different sizes of the square and spiral patterns. Also, in both geometric patterns, the reduction of the adhesive glass width induced C54 to create bonds amongst themselves. NIH 3T3 cells adhered inside the squares and the spiral but reducing the adhesive glass width induced NIH 3T3 to adhere to immobilized Hyal. This fact is explained by the interactions between the cells and the immobilized Hyal as a consequence of the CD44/Hyal binding

Heterotypic cell-cell interaction on micropatterned surfaces

Purpose. The aim of this paper was to study the influence of chemical and topographical signals on cell behaviour and to obtain a heterotypic cell-cell interaction on microstructured domains. Methods. The polysaccharide hyaluronic acid (Hyal) was photoimmobilised on glass surfaces in order to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterised by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The behaviour of Human Coronary Artery Endothelial Cells (HCAEC) and human tumoral dermal fibroblasts (C54) was investigated on these micropatterned surfaces by adhesion studies. Moreover heterotypic interaction among C54 and HCAEC adhered on patterned surfaces was evaluated by time-lapse video microscopy. Results. Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the centre to the edge of the sample. Neither HCAEC nor C54 adhered to the immobilised Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent cells depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain. Conclusions. A heterotypic cell-cell interaction occurred in the same chemical and topographic micro-domains but in narrow areas only. Moreover, the number of cells adhering to the glass domains and cell morphology depended on the dimensions of both adhesive areas and cell nuclei

Chemistry and topographic domains: micropatterned surfaces. Production, physical-chemical and biological characterisation

The presence of micro and nano-domains on a surface allows the manipulation of two fundamental external signals: cellsubstrate and cell-cell interaction, in order to create a pattern of highly oriented cells capable of arranging themselves in tissue. Cell guidance also depends on the different chemical and/or topographic domains present on a surface and on their geometry. Thus, structures are realised so that they also contain different chemical and geometrical domains. Among the many known methods for modifying surfaces, the photoimmobilisation process is a useful technique for creating microstructures with both chemical and topographic patterns. In most approaches, cells are localized to adhesive regions on a substrate, thus limiting their use to one cell type. More recently, approaches have been developed for patterning two or more cell types in spatially defined co-cultures. These approaches can be used to study the effects of cell shape, cell-matrix interactions, and heterotypic cell-cell contact on various cell functions. Many studies on patterned co-cultures have involved the selective adhesion of one cell type compared to the normal adhesion of the other. In this chapter, the results of our research on micro-topography and cell behaviour are reviewed and discussed. Patterns with different geometries have been obtained by photoimmobilisation of the polysaccharide hyaluronic acid (Hyal) on glass surfaces. The resulting surface patterns have been utilised to study the influence of microstructures as a function of chemical, topographic and dimensional properties on both primary and tumoral cell lines. Cell adhesion and distribution have been analysed as a function of the shape and area of the microdomains. Studies carried out with the aim to analyse heterotypic cell-cell interaction as a function of geometry and dimension domains are also reported. In particular, the possibility of obtaining co-cultured microstructured surfaces by seeding fibroblasts on patterned samples with already adhered endothelial cells has been investigated. The chapter ends with an outline of the results recently obtained by other authors studying the combined effect of topographic domains and mechanical/gravitational stress, a very promising topic in the field of cell microenvironment topography

Heterotypic interaction of fibroblasts and endothelial cells on restricted area

The polysaccharide hyaluronic acid (Hyal) was photoimmobilized on glass surfaces to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Attenuated Total Reflection Infrared Spectroscopy (ATR FT-IR), and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the center to the edge of the sample. The behavior of Human Coronary Artery Endothelial Cells (HCAEC) and human tumoral dermal fibroblasts (C54) was studied on these micropatterned surfaces. Neither HCAEC nor C54 adhered to the immobilized Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent HCAEC and C54 depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain for the first time. In comparison to other techniques for patterning two different cell types, our approach was non cytotoxic and allowed arbitrary geometric patterns to form on different biocompatible substrata