| 1. |
- Belak, Sandor, et al.
(författare)
-
Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China
- 2011
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 171, s. 200-205
-
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.
|
|
| 2. |
- Belak, Sandor
(författare)
-
Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus
- 2010
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 167, s. 165-171
-
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
|
|
| 3. |
- Blomström, Anne-Lie, et al.
(författare)
-
Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV)
- 2013
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 189, s. 1-6
-
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.
|
|
| 4. |
|
|
| 5. |
- Fossum, Caroline, et al.
(författare)
-
PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells
- 2014
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 196, s. 185-192
-
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.
|
|
| 6. |
- Hjertner, Bernt, et al.
(författare)
-
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
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 175, s. 149-155
-
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.
|
|
| 7. |
- 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.
|
|
| 8. |
- 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.
|
|
| 9. |
|
|
| 10. |
|
|
