Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.
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.
Foreign Animal Disease Diagnostic Laboratory, National Veterinary Service Laboratory, Veterinary Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, Greenport, NY 11944, USA.
Ming Y
MY
Deng
He
H
Wang
Gordon B
GB
Ward
Tammy R
TR
Beckham
Thomas S
TS
McKenna
D000818:Animals; D006691:Classical Swine Fever / Q000097:blood / Q000175:diagnosis* / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000302:isolation & purification*; D006321:Heart / Q000821:virology; D007668:Kidney / Q000821:virology; D008099:Liver / Q000821:virology; D008198:Lymph Nodes / Q000821:virology; D014066:Palatine Tonsil / Q000821:virology; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000821:virology; D013552:Swine / Q000097:blood / Q000821:virology*; D001743:Urinary Bladder / Q000821:virology
D012367:RNA, Viral