Comparison of four RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus by RT-PCR.
We compared four commercial RNA isolation methods for the extraction of RNA from porcine reproductive and respiratory syndrome virus (PRRSV). The sensitivity of the methods was determined by extraction of RNA from serial 10-fold dilutions of PRRSV diluted in PRRSV-negative porcine serum or in Eagle's minimal essential medium (EMEM) followed by amplification of extracted nucleic acids by RT-PCR. The PCR products were detected in ethidium bromide-stained agarose gels. The QIAamp viral kit, which is based on binding of RNA to silica particles, was the most sensitive, allowing the detection of an equivalent of 10 TCID50 of virus in 100 microliters of serum or EMEM. The QIAamp-tissue and the TRIzol LS kits detected 100 TCID50 per 100 microliters in both diluents whereas Ultraspec-3 detected 100 TCID50 of virus in EMEM and 1000 TCID50 in serum. These results indicate that QIAamp viral kit has a higher sensitivity for RT-PCR analysis of PRRSV.
Guarino
H
H
Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul 55108, USA.
Goyal
S M
SM
Murtaugh
M P
MP
D000818:Animals; D016133:Polymerase Chain Reaction / Q000379:methods; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic / Q000821:virology*; D012680:Sensitivity and Specificity; D013552:Swine
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic