Comparison of methods for improved RNA extraction from blood for early detection of Classical swine fever virus by real-time reverse transcription polymerase chain reaction.

摘要

Classical swine fever (CSF) is a highly contagious disease of pigs. Early detection of the Classical swine fever virus (CSFV) in infected animals and routine surveillance is important for a rapid response and control of an outbreak of CSF. The current study investigated whole blood as a clinical specimen for the detection of CSFV by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in experimentally infected pigs. The virus was detectable in pre-clinical animals in whole blood and in different fractions of blood, including white blood cells, red blood cells (RBC), and serum. Based on an in-vitro binding assay, CSFV is retained in the RBC fraction. Naturally occurring PCR inhibitors of whole blood were shown to inhibit detection, and several commercial RNA extraction kits failed to remove these inhibitors. The commercial blood RNA extraction protocols were modified to achieve optimized single tube and high-throughput 96-well plate RNA extraction that efficiently removed PCR inhibitors from whole blood and enhanced detection of CSFV in experimentally inoculated pigs. This enabled detection 1-2 days earlier than observed using unmodified RNA extraction protocols. The results show effective use of whole blood as a clinical specimen for diagnosis and surveillance of CSF in pre-clinical animals.