| 1. |
- Viazov, Sergei, et al.
(författare)
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Typing of hepatitis C virus isolates by DNA enzyme immunoassay
- 1994
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 48:1, s. 81-91
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Tidskriftsartikel (refereegranskat)abstract
- Recently, at least six types of hepatitis C viruses (HCV) have been identified. Different types of HCV appear to possess different pathogenic properties and a different sensitivity to interferon treatment. Typing of HCV isolates may therefore be an important diagnostic procedure. We report on a new method for identification of HCV types 1a, 1b, 2a, 2b and 3a which are most prevalent in Europe, North America and Japan. The assay is based on a combination of two well established techniques, the polymerase chain reaction (PCR) and DNA enzyme immunoassay (DEIA). In the first step of the method a cDNA of about 250 bp corresponding to the HCV core-region is amplified by nested PCR. The target cDNA is then hybridized to type-specific oligonucleotides fixed to a solid phase through an avidin-biotin bridge. The formed hybrids are detected by a standard ELISA using monoclonal antibodies reacting with double-stranded DNA. Typically, signal-to-noise (S/N) ratios between 18.2 and 48.6 could be observed when different HCV types/subtypes were analyzed by this method. The test was evaluated using cloned HCV cDNAs of known types and by sequence determination of some of the typed cDNAs. Typing of 115 isolates from Germany, Russia and Turkey revealed that subtype 1b (59-100%) and 1a (24-32%) are most prevalent in these countries.
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| 2. |
- Widell, Anders, et al.
(författare)
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A microcarrier cell culture system for large scale production of hepatitis A virus
- 1984
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 8:1-2, s. 63-71
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.
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| 3. |
- Elgh, Fredrik, 1957-, et al.
(författare)
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A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus.
- 1996
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 59:1-2, s. 161-72
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Tidskriftsartikel (refereegranskat)abstract
- Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU). The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man. We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE. Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed. The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies. The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94. A recombinant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE. ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity as compared to the full length recombinant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes. Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions.
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| 4. |
- Forsman, Anna, et al.
(författare)
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Single-tube nested quantitative PCR : a rational and sensitive technique for detection of retroviral DNA. Application to RERV-H/HRV-5 and confirmation of its rabbit origin
- 2003
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Ingår i: Journal of Virological Methods. - 0166-0934 .- 1879-0984. ; 111:1, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- It was reported earlier that a few patients suffering from non-Hodgkin's lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkin's lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkin's lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per microg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkin's lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.
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| 5. |
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| 6. |
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| 7. |
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| 8. |
- 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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| 9. |
- 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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| 10. |
- Belak, Sandor, et al.
(författare)
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Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China
- 2011
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 171, s. 200-205
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Tidskriftsartikel (refereegranskat)abstract
- A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65 degrees C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis. (C) 2010 Elsevier B.V. All rights reserved.
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| 11. |
- Belak, Sandor
(författare)
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Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 167, s. 165-171
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Tidskriftsartikel (refereegranskat)abstract
- A real-nine RT-PCR assay based on the primer-probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well A distinguishing characteristic of the assay is its tolerance toward mutations in the probe region. Furthermore, melting curve analysis following immediately PCR confirms specific probe hybridization and can reveal mutations in the probe region by showing a difference in the melting point The assay sensitivity was in the range of 10-100 target copies and the specificity tests showed no positive results for heterologous pathogens The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008 The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies The assay sensitivity for some other serotypes that circulate currently in Europe was also determined BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging strains (C) 2010 Elsevier B V. All rights reserved
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| 12. |
- 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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| 13. |
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| 14. |
- Blomström, Anne-Lie, et al.
(författare)
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Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV)
- 2013
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 189, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan (R) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus. (C) 2013 Elsevier B.V. All rights reserved.
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| 15. |
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| 16. |
- El Bagoury, Gabr F., et al.
(författare)
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Development and evaluation of one-step real-time RT-PCR assay for improved detection of foot-and-mouth disease virus serotypes circulating in Egypt
- 2022
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Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 306
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Tidskriftsartikel (refereegranskat)abstract
- Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RTqPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.
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| 17. |
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| 18. |
- 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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| 19. |
- Forsgren, Eva, et al.
(författare)
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Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization
- 2017
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 246, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at -80 degrees C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24 h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder (TM) homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15 min.
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| 20. |
- Forsgren, Eva
(författare)
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Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study
- 2017
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 248, s. 217-225
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Tidskriftsartikel (refereegranskat)abstract
- The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log(10) CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log(10) CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n = 270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (S-R) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log(10) CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/- 1.96 S-R) at the diagnostic threshold (8 log(10) CBPV/bee) was +/- 2.08 log(10) CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log(10) CBPV/bee may be considered to indicate probable cases of chronic paralysis.
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| 21. |
- 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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| 22. |
- Fossum, Caroline, et al.
(författare)
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PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells
- 2014
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 196, s. 185-192
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Tidskriftsartikel (refereegranskat)abstract
- A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS). Peripheral blood mononuclear cells (PBMCs) obtained from a pig with severe PMWS produced PCV2-SPOT5 spontaneously whereas PBMCs obtained from pigs infected subclinically only generated PCV2-SPOT5 upon in vitro stimulation. The PCV2 secretion potential was related to the PCV2 DNA content in the PBMCs as determined by two PCV2 real-time PCR assays, developed to differentiate between Swedish PCV2 genogroups 1 (PCV2a) and 3 (PCV2b). Besides the current application these qPCRs could simplify future epidemiological studies and allow genogroup detection/quantitation in dual infection experiments and similar studies. The developed PCV2-SPOT assay offers a semi-quantitative approach to evaluate the potential of PCV2-infected porcine cells to release PCV2 viral particles as well as a system to evaluate the ability of different cell types or compounds to affect PCV2 replication and secretion. (C) 2013 Elsevier B.V. All rights reserved.
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| 23. |
- 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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| 24. |
- 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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| 25. |
- 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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| 26. |
- Hjertner, Bernt, et al.
(författare)
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Development and comparison of a Primer-Probe Energy Transfer based assay and a 5 ' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus
- 2011
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 175, s. 149-155
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Tidskriftsartikel (refereegranskat)abstract
- In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 x 10(8) to 2 x 10(2) copies/mu l. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV. (C) 2011 Elsevier B.V. All rights reserved.
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| 27. |
- Hjertner, Bernt, et al.
(författare)
-
Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay
- 2011
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 174, s. 117-119
-
Tidskriftsartikel (refereegranskat)abstract
- A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5′ conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2×101 to 2×108 copies and amplification time was approximately 2h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5′ untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications.
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| 28. |
- Hornyak, Akos, et al.
(författare)
-
Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)
- 2012
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 181:2, s. 155-163
-
Tidskriftsartikel (refereegranskat)abstract
- Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Kaliff, Malin, 1985-, et al.
(författare)
-
Optimization of droplet digital PCR assays for the type specific detection and quantification of five HPV genotypes, also including additional data on viral load of nine different HPV genotypes in cervical carcinomas
- 2021
-
Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 294
-
Tidskriftsartikel (refereegranskat)abstract
- The droplet digital PCR (ddPCR) system enables high-sensitivity detection of nucleic acids and direct absolute quantification of the targets. The aim of this research was to evaluate this system for viral load (VL) analysis of the human papillomavirus (HPV) genotypes HPV31, 35, 39, 51 and 56 measured in number of viral particles per cell. The sample types used for the optimization of the ddPCR assay were formalin-fixed paraffin-embedded (FFPE) tissues and cervical liquid cytology samples. The presently optimized ddPCR assays, together with assays optimized previously for HPV16, 18, 33 and 45, with the same ddPCR method, were used for the VL analysis of cervical tumor samples. Results published previously on the present study cohort showed that women with a cervical tumor containing multiple high-risk HPV genotypes had a worse prognosis compared to women with single-genotype-infected tumors. The VL was therefore analyzed in this study for the same cohort, as a possible explanatory factor to the prognostic differences. The results of the optimization part of the study, with analysis of VL using ddPCR in DNA from varying sample types (FFPE and liquid cytology samples), showed that each of the five assays demonstrated good inter- and intra-assay means with a coefficient of variation (CV) under 8% and 6% respectably. The cohort results showed no difference in VL between tumors with multiple and single HPV infections, and therefore did most likely not constitute a contributing factor for prognostic differences observed previously. However, tumors from women aged 60 years or older or containing certain HPV genotypes and genotype genera were associated with a higher VL.
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| 33. |
- Kallies, Rene, et al.
(författare)
-
Development and characterization of murine monoclonal antibodies to first and second Ljungan virus genotypes
- 2012
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 184:1-2, s. 27-33
-
Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV) is a rodent pathogen that causes diabetes and myocarditis in its natural host. In addition, LV has been associated with human disease during pregnancy and of neonates, respectively. A panel of 22 monoclonal antibodies (mAbs) against first and second LV genotypes were produced by immunization of BALB/c mice with whole virus. Thirteen mAbs were class IgG antibodies and nine were class IgM antibodies; all of them contained kappa light chains. All mAbs were reactive with LV by capture enzyme-linked immunosorbent assay and indirect immunofluorescence assay. In addition, five mAbs showed a positive staining in immunohistochemistry. No mAb bound to denatured capsid proteins detected by western immunoblotting. In contrast, the target capsid protein(s) of 20 mAbs were identified by immune precipitation, revealing the conformational nature of epitopes required for mAb binding. None of the mAbs reacted with third and fourth LV genotypes. mAbs characterized should provide useful tools for the development of diagnostic assays and the investigation of LV first and second genotype properties and its pathogenesis.
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| 34. |
- 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
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 144:1-2, s. 27-31
-
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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| 35. |
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| 36. |
- Käller, Max, et al.
(författare)
-
Tag-array based HPV genotyping by competitive hybridization and extension
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 129:2, s. 102-112
-
Tidskriftsartikel (refereegranskat)abstract
- A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (< 2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.
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| 37. |
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| 38. |
- Larska, Magdalena, et al.
(författare)
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Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus
- 2013
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 187, s. 103-109
-
Tidskriftsartikel (refereegranskat)abstract
- Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental infection of calves with atypical pestivirus, BVDV-1 and a combination of both viruses have been examined by routine and new diagnostic tests to validate their robustness and sensitivity. As expected, virus neutralization tests using homologous virus were able to differentiate the two groups infected by BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect antibodies also from cattle infected with atypical pestivirus a few weeks after infection. The detection, however, was at a lower level and slightly delayed. Statistical validation of the threshold suggested by the manufacturer showed that in some cases the reduction of the cut-off values would improve the test sensitivity. (C) 2012 Elsevier B.V. All rights reserved.
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| 39. |
- Leblanc, Neil, et al.
(författare)
-
Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
- 2009
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155:1, s. 1-9
-
Tidskriftsartikel (refereegranskat)abstract
- This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.
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| 40. |
- Lillsunde Larsson, Gabriella, 1971-, et al.
(författare)
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HPV Genotyping from the high risk mRNA Aptima assay : a direct approach using DNA from Aptima sample tubes
- 2016
-
Ingår i: Journal of Virological Methods. - Amsterdam, Netherlands : Elsevier. - 0166-0934 .- 1879-0984. ; 235, s. 80-84
-
Tidskriftsartikel (refereegranskat)abstract
- The underlying cause of cervical cancer is infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.
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| 41. |
- Lindahl, Johanna, et al.
(författare)
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A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya
- 2019
-
Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 269, s. 70-76
-
Tidskriftsartikel (refereegranskat)abstract
- Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens.
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| 42. |
- Liu, Lihong, et al.
(författare)
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Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus
- 2009
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 160, s. 69-73
-
Tidskriftsartikel (refereegranskat)abstract
- Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF. (C) 2009 Elsevier B.V. All rights reserved.
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| 43. |
- Liu, Lihong, et al.
(författare)
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Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars
- 2009
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 159, s. 131-133
-
Tidskriftsartikel (refereegranskat)abstract
- Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a disease notifiable to the Office international des Epizooties (OIE). A live marker vaccine would be the ultimate choice for controlling CSF, which enables serological and genetic differentiation of vaccine from wild type CSFV. Recently, a marker vaccine CP7_E2alf has been reported [Koenig, P., Lange, E., Reimann, L, Beer, M., 2007. CP7_E2alf. a safe and efficient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV). Vaccine 25, 3391-3399]. A vaccine-specific TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-specific assay was modified from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run. The detection limit is 50 viral RNA copies per reaction for the vaccine-specific assay, and 20 copies per reaction for the wild type assay. The two assays have been shown to be highly specific and reproducible, with potential application for genetic differentiation of wild type CSFV from the marker vaccine CP7_E2alf in wild boar vaccination programs. (C) 2009 Elsevier B.V. All rights reserved.
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| 44. |
- Liu, Lihong, et al.
(författare)
-
Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus
- 2010
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 167, s. 74-78
-
Tidskriftsartikel (refereegranskat)abstract
- Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method applied and adapted to the detection of a number of pathogens. A LAMP assay was developed for the specific detection of wild-type classical swine fever virus (CSFV). Based on an alignment of genomic sequences of pestiviruses available in GenBank, four primers were selected targeting six positions in the NS5B gene region of the viral genome. The assay was performed in a simple water bath at a constant temperature of 62 degrees C, and after adding SYBR Green I, the results were visualised by the naked eye. This assay allowed easy applicability in a laboratory that is equipped simply, or even on site, close to the outbreaks and under field conditions. For confirmation, the results could also be visualised under UV light or by separation of the amplified products on 2% agarose gels. The detection limit of the assay was 2.5 medium tissue culture infectious dose (TCID(50)). The assay was validated with 116 clinical samples from vaccinated swineherds and 53 blood samples from experimental infections, and the results were comparable to a real-time RTPCR assay. In summary, the LAMP assay provides a simple, rapid, and sensitive tool for the detection of wild-type CSFV under field conditions. (C) 2010 Elsevier B.V. All rights reserved.
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| 45. |
- Mantke, Oliver Donoso, et al.
(författare)
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A new quantitative real-time reverse transcriptase PCR assay and melting curve analysis for detection and genotyping of Ljungan virus strains
- 2007
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 141:1, s. 71-77
-
Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV), a new member of the Picornaviridoe, recently isolated from vole species in both Sweden and the USA, is suspected to be pathogenic for humans as an actiological agent in myocarditis, diabetes, neurological disease and perinatal disease. This study describes for the first time an RT-PCR assay that can identify and quantify LV infection in various tissue sample types using primers and two different minor-groove-binder probes targeting the 5'-untranslated region of the LV genome. The assay, evaluated using control samples derived from various virus cultures and rodent tissues, allows precise quantification of viral load over six orders of magnitude (101 to 106 viral copies per assay) for all known strains of LV with high sensitivity and specificity. Furthermore, a melting curve analysis (MCA) was developed using two amplicon-specific hybridisation probes that allows rapid genotyping of different LV strains. These new methods provide useful tools to investigate the putative role of LV as a pathogen in both rodents and humans.
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| 46. |
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| 47. |
- Mujtaba, Tahir
(författare)
-
Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex
- 2020
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 276
-
Tidskriftsartikel (refereegranskat)abstract
- Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield. The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.
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| 48. |
- Muradrasoli, Shaman, et al.
(författare)
-
Broadly targeted multiprobe QPCR for detection of coronaviruses : Coronavirus is common among mallard ducks (Anas platyrhynchos)
- 2009
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 159:2, s. 277-287
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Tidskriftsartikel (refereegranskat)abstract
- Coronaviruses (CoV) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan((R))) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in seven of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.
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| 49. |
- Muradrasoli, Shaman, et al.
(författare)
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Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy
- 2010
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 163:2, s. 313-322
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Tidskriftsartikel (refereegranskat)abstract
- A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan (R)-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon: MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan (R) probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan (R) probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.
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| 50. |
- Muradrasoli, Shaman, et al.
(författare)
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Development of real-time PCRs for detection and quantitation of human MMTV-Iike (HML) sequences HML expression in human tissues
- 2006
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 136:1-2, s. 83-92
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Tidskriftsartikel (refereegranskat)abstract
- The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1 - 10, also referred to as human endogenous retroviruts "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan((R))-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML 1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.
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