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.
Deng
Ming Y
MY
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.
Wang
He
H
Ward
Gordon B
GB
Beckham
Tammy R
TR
McKenna
Thomas S
TS
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