Detection and quantitative pathogenesis study of classical swine fever virus using a real time RT-PCR assay.
A real time reverse transcription (RT) TaqMan PCR assay for the detection of classical swine fever virus (CSFV) previously described for use on a SmartCycler was validated on the Applied Biosystems AB 7700 Sequence Detection System using the Roche MagNA pure instrument for nucleic acid extraction and reaction set up. The primers and probe were specific for the CSFV strains (NSW, Baker and Weybridge) and did not react with other pestiviruses (BDV Tobias, BDV #327, BVDV non-CPE and BVDV C24V). Analysis of blood samples collected from pigs 1-6 and 8 days post-oronasal infection showed that over >10(6) range there was a linear relationship between log10TCID50ml-1 blood and the log10 normalised genetic load measured by quantitative TaqMan assay. The assay was used to assess CSFV shedding from infected pigs by quantitative TaqMan assay of virus genetic loads in tonsil, nasal and rectal swabs. Infection of tonsils was detected as early as 1 day post-inoculation. Shedding of virus detected by nasal and rectal swabs commenced on the third day post-inoculation. Quantitative TaqMan was used to analyse virus genetic load in tissues collected from pigs killed on days 1-3, 5 and 8 post-infection. Virus infection appeared first in tonsil (day 1), then submandibular lymph node, spleen, ileum and mesenteric lymph node (by day 3). Thereafter, virus spread to the visceral organs and finally to the pancreas and brain. Tonsil, nasal and rectal swabs as well as whole blood were found to be suitable samples for the rapid detection of CSFV using the TaqMan assay and automated nucleic acid extraction and reaction set up.
Ophuis
Ralph J A Oude
RJ
CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag 24, Geelong, Vic. 3220, Australia.
Morrissy
Chris J
CJ
Boyle
David B
DB
D000818:Animals; D001483:Base Sequence; D006691:Classical Swine Fever / Q000175:diagnosis* / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000821:virology; D014066:Palatine Tonsil / Q000821:virology; D012007:Rectum / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D013552:Swine; D013997:Time Factors
D017931:DNA Primers