A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan)
| Autor: | Georgina Ibata, J. P. Lowings, A McGoldrick, Sándor Belák, J. J. Sands, D.J Paton |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Time Factors
Swine Cell Culture Techniques Enzyme-Linked Immunosorbent Assay Kidney Polymerase Chain Reaction Sensitivity and Specificity law.invention Classical Swine Fever law Virology TaqMan Animals Polymerase chain reaction biology Age Factors Amplicon biology.organism_classification Molecular biology Reverse transcriptase Reverse transcription polymerase chain reaction Real-time polymerase chain reaction Classical swine fever Classical Swine Fever Virus RNA Viral Nested polymerase chain reaction |
| Zdroj: | Journal of virological methods. 72(2) |
| ISSN: | 0166-0934 |
| Popis: | Detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation (VI) in cell cultures, antigen detection, or molecular analysis. To simplify the latter, a 5′-nuclease assay (TaqMan) was developed for the rapid and specific detection of CSFV with the minimum of downstream PCR processing. A pair of 5′-non-coding region, panpestivirus-specific PCR primers were assessed in a one-step reverse transcription-PCR with each of 36 diverse pestiviruses. The PCR products were subsequently reamplified, in conjunction with a CSFV-specific fluorogenic probe, in a nested-PCR with a second set of panpestivirus PCR primers. During nested PCR, when the target of interest was present, the CSFV probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5′-3′ nucleolytic activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. Each PCR tube was then placed directly into a luminescence spectrometer to monitor for any increase in fluorescence due to cleavage of the probe. This assay detected representatives of all genetic sub-groups of CSFV, but gave negative results for other pestiviruses. A preliminary assessment showed that the method could be used to detect CSFV RNA extracted from infected pig blood with a sensitivity greater than that of VI. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect