Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR

J Vet Diagn Invest. 2005 Nov;17(6):574-8. doi: 10.1177/104063870501700609.

Abstract

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding beta-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Classical Swine Fever / blood
  • Classical Swine Fever / diagnosis*
  • Classical Swine Fever / virology*
  • Classical Swine Fever Virus / genetics*
  • Classical Swine Fever Virus / isolation & purification*
  • Heart / virology
  • Kidney / virology
  • Liver / virology
  • Lymph Nodes / virology
  • Palatine Tonsil / virology
  • RNA, Viral / blood*
  • RNA, Viral / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Spleen / virology
  • Swine / blood
  • Swine / virology*
  • Urinary Bladder / virology

Substances

  • RNA, Viral