1,721,121 research outputs found
Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus
No full textTo optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (JPC) concentration on the quantitative results of HSV1. HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1 HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays. (C) 2008 Elsevier B.V. All rights reserved
Confusions in orbivirus protein classification
Full text linkAn extensive comparative analysis of orbivirus genomes revealed four cases of unclear numeration and protein designation, due to confused reference to protein size or segment size by which they are encoded. A concise nomenclature based on type species, sequence homology and functional characteristics independent of segment or protein size is suggested
A phaseguided passive batch microfluidic mixing chamber for isothermal amplification
No full textWith a view to developing a rapid pathogen detection system utilizing isothermal nucleic acid amplification, the necessary micro-mixing step is innovatively implemented on a chip. Passive laminar flow mixing of two 6.5 mu l batches differing in viscosity is performed within a microfluidic chamber. This is achieved with a novel chip space-saving phaseguide design which allows, for the first time, the complete integration of a passive mixing structure into a target chamber. Sequential filling of batches prior to mixing is demonstrated. Simulation predicts a reduction of diffusive mixing time from hours down to one minute. A simple and low-cost fabrication method is used which combines dry film resist technology and direct wafer bonding. Finally, an isothermal nucleic acid detection assay is successfully implemented where fluorescence results are measured directly from the chip after a one minute mixing sequence. In combination with our previous work, this opens up the way towards a fully integrated pathogen detection system in a lab-on-a-chip format
Complete Genome Sequence of Tick-Borne Encephalitis Virus Strain A104 Isolated from a Yellow-Necked Mouse (Apodemus flavicollis) in Austria.
No full textTick-borne encephalitis virus (TBEV) strain A104 was isolated from the brain of a yellow-necked mouse in Austria in 1990. The complete genome sequence was 11,097 nucleotides long. Comparison with TBEV prototype strain Neudoerfl showed 32 amino acid exchanges and the absence of an internal poly(A) stretch within the 3' noncoding region
Interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells
No full textSevere acute respiratory syndrome (SARS) of humans is caused by a novel coronavirus of zoonotic origin termed SARS-associated coronavirus (SARS-CoV). The virus induces severe injury of lung tissue, as well as lymphopenia. and destruction of the architecture of lymphatic tissue by as-yet-unknown mechanisms. In this study, the interaction of SAIRS-CoV with dendritic cells (DCs), the key regulators of immune responses, was analysed. Monocyte-clerived DCs were infected with SARS-CoV and analysed for viability, surface-marker expression and alpha interferon (IFN-alpha) induction. SAIRS-CoV infection was monitored by quantitative RT-PCIR, immunofluorescence analysis and recovery experiments. SARS-CoV infected both immature and mature DCs, although replication efficiency was low. Immature DiCs were activated by SARS-CoV infection and by UV-inactivated SARS-CoV. Infected DCs were still viable on day 6 post-infection, but major histocompatibility complex class I upregulation was missing, indicating that DC function was impaired. Additionally, SARS-CoV infection induced a delayed activation of IFN-alpha expression. Therefore, it is concluded that SARS-CoV has the ability to circumvent both the innate and the adaptive immune systems
Tick-borne encephalitis virus in Clethrionomys glareolus in the Czech Republic
No full textA total of 474 specimens from 157 rodents caught at the military training area of Boletice in the south of the Czech Republic from May to November 1999 were screened for TBEV by nested PCR. TBEV-specific RNA was amplified from lung, kidney, and spleen derived from one Clethrionomys glareolus in the first RT-PCR round. Sequence analysis revealed a 100% identity to the TBEV strain Neudoerfl. TBEV presence in the sample was confirmed by mouse brain passage of backup samples and cell culture. The results support the observation that hantaviruses and TBEV transmission can occur sympatrically in the same rodent population
Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification
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