A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan).
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.
Central Veterinary Laboratory, Veterinary Laboratories Agency, Addlestone, Surrey, UK.
A
A
McGoldrick
J P
JP
Lowings
G
G
Ibata
J J
JJ
Sands
S
S
Belak
D J
DJ
Paton
D000367:Age Factors; D000818:Animals; D018929:Cell Culture Techniques; D006691:Classical Swine Fever / Q000097:blood / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D007668:Kidney; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012367:RNA, Viral / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013552:Swine / Q000097:blood / Q000821:virology*; D013997:Time Factors
D012367:RNA, Viral