Format

Send to

Choose Destination
See comment in PubMed Commons below
J Virol Methods. 2005 Dec;130(1-2):36-44. Epub 2005 Aug 1.

Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever.

Author information

1
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Boddenblick 5a, D-17493 Greifswald-Insel Riems, Germany.

Abstract

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.

PMID:
16055202
DOI:
10.1016/j.jviromet.2005.05.030
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center