| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
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| 5. |
- 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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| 6. |
- 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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| 7. |
- Hjertner, Bernt, et al.
(författare)
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Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay
- 2011
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 174, s. 117-119
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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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| 8. |
- Hornyak, Akos, et al.
(författare)
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Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)
- 2012
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 181:2, s. 155-163
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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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| 9. |
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| 10. |
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| 11. |
- Kallies, Rene, et al.
(författare)
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Development and characterization of murine monoclonal antibodies to first and second Ljungan virus genotypes
- 2012
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 184:1-2, s. 27-33
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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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| 12. |
- 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
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 187, s. 103-109
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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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| 13. |
- Liu, Lihong, et al.
(författare)
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Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 167, s. 74-78
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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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| 14. |
- 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
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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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| 15. |
- Nielsen, Alex Christian Yde, et al.
(författare)
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A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience
- 2013
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 193:2, s. 359-363
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Tidskriftsartikel (refereegranskat)abstract
- As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66(3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms. (C) 2013 Elsevier B.V. All rights reserved.
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| 16. |
- Näslund, Jonas, 1979-, et al.
(författare)
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Detection of Puumala and Rift Valley Fever virus by quantitative RT-PCR and virus viability tests in samples of blood dried and stored on filter paper
- 2011
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Ingår i: Journal of Virological Methods. - Amsterdam : Elsevier. - 0166-0934 .- 1879-0984. ; 178:1-2, s. 186-90
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Tidskriftsartikel (refereegranskat)abstract
- Haemorrhagic fever viruses cause emerging infections worldwide, and blood or serum is the main sample used for diagnosis. However, storage and transportation of such samples from remote areas to regional laboratories may be complicated and expensive. In this study, a novel approach was evaluated for the detection of Puumala hantavirus (PUUV) RNA and Rift Valley fever virus (RVFV) RNA. Whole-blood samples spiked with viable virus particles were tested in parallel with clinical samples from patients with acute haemorrhagic fever with renal syndrome (nephropathia epidemica). Individual blood samples were spotted on filter paper, dried, and used for RNA extraction at later time points. PUUV RNA was detected by RT-PCR after storage at room temperature for up to six weeks. In contrast, only low copy numbers of RVFV RNA were detected after 1-2 days even though viable RVFV was eluted from the dried filter papers after the same time. The use of filter paper to collect and store blood samples for PUUV RNA detection is therefore a simple and reliable procedure. This approach might facilitate sampling and analysis of other RNA viruses from human or animal sources and could be used for field studies in remote areas or in developing countries.
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| 17. |
- Orozovic, Goran, 1965-, et al.
(författare)
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Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 170:1-2, s. 94-98
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1-9). A total of 11 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny.
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| 18. |
- Rodrigues De Miranda, Joachim
(författare)
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Development and validation of a real-time two-step RT-qPCR TaqMan (R) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies
- 2014
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 197, s. 7-13
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Tidskriftsartikel (refereegranskat)abstract
- Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen subcuticular pouches, forming the characteristic fluid-filled sac that gives its name to the disease. Outbreaks of the disease have been reported in different countries, affecting the development of the brood and causing losses in honey bee colonies. Today, few data are available on the SBV viral load in the case of overt disease in larvae, or for the behavioural changes of SBV-infected adult bees. A two-step real-time RT-PCR assay, based on TaqMan (R) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued by the French Standards Institute, where the reliability and the repeatability of the results and the performance of the assay were confirmed. The performance of the qPCR assay was validated over the 6 log range of the standard curve (i.e. from 10(2) to 10(8) copies per well) with a measurement uncertainty evaluated at 0.11 log(10). The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5 mu l of cDNA. The RT-qPCR assay was applied during a French SBV outbreak in 2012 where larvae with typical SBV signs were collected, along with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome copies per individual). (C) 2013 Elsevier B.V. All rights reserved.
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| 19. |
- Sauerbrei, A., et al.
(författare)
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Screening of herpes simplex virus type 1 isolates for acyclovir resistance using DiviTum® assay
- 2013
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 188:1-2, s. 70-72
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Tidskriftsartikel (refereegranskat)abstract
- Rapid alternative methods are required to evaluate easily acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates. The objective of this study was to screen 54 ACV-sensitive and 41 ACV-resistant clinical HSV-1 isolates, well characterized by phenotypic and genotypic methods, for the phosphorylation activity of the viral thymidine kinase (TK) using a commercially available and modified non-radioactive DiviTum® test on the basis of an indirect enzyme linked immunosorbent assay. The ACV-sensitive HSV-1 isolates had high TK activity values between 31.5±6.4 DiviTum® Units per liter (DU/L) and 487.4±60.1DU/L. The mean activity of all ACV-sensitive isolates was calculated as 212.3±15.7 DU/L. By contrast, the mean activity of all ACV-resistant HSV-1 isolates was significantly lower at 5.5±1.3DU/L. Out of the 41 ACV-resistant HSV-1 isolates, 38 had no or very low phosphorylation activities of the viral TK between 0DU/L and 9.3±3.2DU/L. The remaining three ACV-resistant viral isolates had TK activities between 44.6±5.1DU/L and 80.9±13.3DU/L. In conclusion, the modified DiviTum® test can be used to screen HSV-1 isolates for their sensitivity to ACV. Acyclovir-sensitive HSV-1 isolates show TK activities >30DU/L and ACV-resistant isolates have activity values <10DU/L. However, single ACV-resistant HSV-1 isolates can have TK activity values >30DU/L. These strains are most likely ACV-resistant TK-altered mutants, but no evidence was provided for an alteration of the TK.
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| 20. |
- Schlingemann, Joerg, et al.
(författare)
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Novel means of viral antigen identification : Improved detection of avian influenza viruses by proximity ligation
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 163:1, s. 116-122
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Tidskriftsartikel (refereegranskat)abstract
- Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study. the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safety can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.
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| 21. |
- Ståhl, Karl, et al.
(författare)
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Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus
- 2011
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 172, s. 8-15
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Tidskriftsartikel (refereegranskat)abstract
- African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10(9) to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10(-1) to 10(-5). The detection limit was 10(-5) dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field. (C) 2010 Elsevier B.V. All rights reserved.
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| 22. |
- Thomsson, Elisabeth, 1975, et al.
(författare)
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Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology.
- 2011
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Ingår i: Journal of virological methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 175:1, s. 53-9
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Tidskriftsartikel (refereegranskat)abstract
- A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.
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| 23. |
- Vijayaraghavan, Balaje, et al.
(författare)
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Evaluation of envelope glycoprotein E-rns of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus
- 2012
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 185, s. 193-198
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Tidskriftsartikel (refereegranskat)abstract
- Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E-rns of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E-rns protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way. (C) 2012 Elsevier B.V. All rights reserved.
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| 24. |
- Xia, Hongyan, et al.
(författare)
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A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever
- 2011
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 175, s. 170-174
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Tidskriftsartikel (refereegranskat)abstract
- Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N-pro gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV. (C) 2011 Elsevier B.V. All rights reserved.
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| 25. |
- Xia, Hongyan, et al.
(författare)
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A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 168:1-2, s. 18-21
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Tidskriftsartikel (refereegranskat)abstract
- This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The intra- and inter-assay variability are 4.9% and less than 7%, respectively, and variability of bead conjugations is less than 6.6%. The diagnostic performance of the assay was evaluated by testing a total of 509 serum samples. Based on a negative/positive cut-off value of 30.3%, the assay has a sensitivity of 99.4% and a specificity of 98.3% relative to ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control programmes.
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| 26. |
- Xia, Hongyan, et al.
(författare)
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Evaluation of a primer-probe energy transfer real-time PCR assay for detection of classical swine fever virus
- 2010
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 168, s. 259-261
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Tidskriftsartikel (refereegranskat)abstract
- This study describes evaluation of a real-time PCR assay based on primer-probe energy transfer (PriProET) technology for detection of classical swine fever virus (CSFV). The PriProET technology allows melting curve analysis following PCR amplification and thus provides a higher specificity. The assay was compared with a TaqMan assay by testing a total of 203 samples including 175 clinical specimens and 28 batches of Hog Cholera Lapinized Virus (HCLV) vaccine. The two assays gave the same results for 184 (91%) samples. Compared with the TaqMan assay, 19 additional samples were found to be positive for CSFV using the PriProET assay. In an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve: either a wild type-like or a vaccine-like depending on the dominating RNA. The PriProET assay can be a routine molecular tool or a confirmative tool for diagnosis of classical swine fever (CSF), especially in the case of samples that yield an inconclusive result by the TaqMan assay. (C) 2010 Elsevier B.V. All rights reserved.
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