| 1. |
- Ban, L., et al.
(författare)
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An improved detection method for the Rhopalosiphum padi virus (RhPV) allows monitoring of its presence in aphids and movement within plants
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 142:1-2, s. 136-142
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Tidskriftsartikel (refereegranskat)abstract
- Rhopalosiphumpadi virus (RhPV) is an insect RNA virus that infects aphids, reducing their lifespan and fecundity. It can be transmitted vertically between aphids and horizontally via the plant. An improved detection method for the virus in aphids and plants using RT-PCR was developed; this allowed individual aphids to be tested for RhPV. Testing of R. padi aphids collected from different sites in Sweden revealed the presence of RhPV in wild aphid populations for the first time in Europe. Virus could be detected in several life stages of R. padi, including sexual individuals and eggs, establishing an over-wintering route for the virus. Using RT-PCR, systemic transport of the virus in plants was tracked. Virus spread from the aphid feeding site to all parts of the plant, including roots, within 7 days, and could be acquired by virus-free aphids feeding on the same plant.
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| 2. |
- Banér, Johan, et al.
(författare)
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Microarray-based molecular detection of foot-and-mouth disease, vesicular stomatitis and swine vesicular disease viruses, using padlock probes
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 143:2, s. 200-206
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Tidskriftsartikel (refereegranskat)abstract
- The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.
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| 3. |
- Belak, Sandor
(författare)
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Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155, s. 87-90
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Tidskriftsartikel (refereegranskat)abstract
- Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance. (c) 2008 Elsevier B.V. All rights reserved.
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| 4. |
- Formiga-Cruz, Meritxell, et al.
(författare)
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Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 125:2, s. 111-8
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Tidskriftsartikel (refereegranskat)abstract
- Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.
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| 5. |
- Forsman, Anna, et al.
(författare)
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Development of broadly targeted human endogenous gammaretroviralpol-based real time PCRs Quantitation of RNA expression in human tissues
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 129:1, s. 16-30
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Tidskriftsartikel (refereegranskat)abstract
- Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan) and probe-less (SYBR Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created.
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| 6. |
- Fridholm, Helena, et al.
(författare)
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Rapid and reproducible infectivity end-point titration of virulent phage in a microplate system
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 128:1-2, s. 67-71
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Tidskriftsartikel (refereegranskat)abstract
- The standard method for measuring the it umber of infectious phages in solution has traditionally been the plaque forming assay. An alternative method is described where the number of lytic, infectious phages is determined in an endpoint titration assay adapted for a microplate system. In this model system, susceptible Escherichia coli 136 at a density of 4 x 10(7) cells/ml, were mixed with an equal volume (100 mu l) of Phi X174 diluted serially in a microtest plate. After 3 h of incubation on a microplate shaker the endpoint was determined spectrophotornetrically and calculated according to the method of Reed and Muench. A well was considered positive for infection if the OD630-value was <= 10% compared to the OD630-value of the negative control Of uninfected cells. ID50-titers were 2.5 x higher than the PFU-titers (CV 15%) and the intra assay reproducibility revealed a CV of 9%. The method has several advantages as compared with the conventional PFU-titration. It is less time and material consuming with the possibility to assess several samples at the same time.
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| 7. |
- Gyarmati, Péter, et al.
(författare)
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Universal detection of hepatitis E virus by two real-time PCR assays : TaqMan((R)) and Primer-Probe Energy Transfer
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 146:1-2, s. 226-235
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan (R) and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan (R) assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.
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| 8. |
- Hermening, Stephan, et al.
(författare)
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Improved high-capacity adenoviral vectors for high-level neuron-restricted gene transfer to the CNS
- 2006
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 136:1-2, s. 30-37
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Tidskriftsartikel (refereegranskat)abstract
- Adenovirus-based (Ad) vectors are used widely for experimental gene transfer to the CNS. Ad transduce many cell types including postmitotic neurons. However, their use for, CNS gene transfer is limited due to the host immune response elicited. Furthermore, the extensive distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows adenoviral vectors to infect a broad range of host cells which may be disadvantageous in tissues with various different cell types, like the CNS. The use of tissue-specific promoters allows for neuron-restricted gene expression, even though gene expression driven by these promoters is often very weak. Accordingly, increased transgene expression levels from viral transcription units are needed in order to improve the overall performance of Ad vectors. We designed a high-capacity Ad vector (HC-Ad) that allows for high-level, neuron-restricted transgene expression and shows no obvious signs of immumogenicity or toxicity in the mouse brain. (c) 2006 Elsevier B.V. All rights reserved.
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| 9. |
- Karlsson, Malin, et al.
(författare)
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A real-time PCR assay for the monitoring of influenza a virus in wild birds
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 144:1-2, s. 27-31
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Tidskriftsartikel (refereegranskat)abstract
- A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a specific probe. A total of 45 fecal bird samples were analysed for influenza A virus in three different PCR reactions. Overall, 26 samples were positive in at least one of the three real-time PCR assays. Of the 26 samples, 18 were positive by all three reactions. Eight samples were found positive exclusively by the two SYBR green reactions, six of which were detected by both SYBR green reactions. Of the 26 positive samples, 15 samples were verified as positive either by virus isolation or influenza A M2-gene PCR. The results showed that the two SYBR green systems had a higher performance regarding the detection of influenza A as compared to the PCR reaction using a specific probe.
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| 10. |
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