Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever.
A fully validated, ready-to-use, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, multiplexed for simultaneous detection of an internal control, for the simple and rapid diagnosis of classical swine fever (CSF) was developed. Primers and FAM-labeled TaqMan-probes specific for classical swine fever virus (CSFV) were selected from the consensus sequence of the 5' non-translated region (5' NTR) of 78 different CSFV strains. For determining analytical sensitivity, an in vitro transcript (T7-PC3alf) of the 5' NTR was constructed and tested. In addition, the T7-PC3alf transcript was further used as a positive control and a standard for quantitation of CSFV genome copies. A second heterologous in vitro transcript based on a specific primer-probe HEX-system was designed as an internal positive control for the RNA isolation step and RT-PCR. By using limited primer concentrations for the internal control, no adverse effects on the sensitivity of the CSF-system could be observed, and the newly designed duplex real-time RT-PCR proved to have a sensitivity of approximately eight copies. The primer-probe combination selected was strictly CSFV-specific and no amplification was observed in all non-CSFV pestiviruses tested.
Hoffmann
B
B
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Boddenblick 5a, D-17493 Greifswald-Insel Riems, Germany.
Beer
M
M
Schelp
C
C
Schirrmeier
H
H
Depner
K
K
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006691:Classical Swine Fever / Q000175:diagnosis* / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
D020121:5' Untranslated Regions; D017931:DNA Primers