title: Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens.
abstract: The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.
authors.affiliation: Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, 1600 South 16th Street, Ames, IA 50011-1250, USA. wchittic@iastate.edu
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Chittick
authors.forename: Wendy R
authors.initials: WR
authors.lastname: Stensland
authors.forename: John R
authors.initials: JR
authors.lastname: Prickett
authors.forename: Erin L
authors.initials: EL
authors.lastname: Strait
authors.forename: Karen
authors.initials: K
authors.lastname: Harmon
authors.forename: Kyoung-Jin
authors.initials: KJ
authors.lastname: Yoon
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Zimmerman
mesh_terms: D000818:Animals; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012463:Saliva / Q000821:virology*; D013552:Swine
chemical_list: D012367:RNA, Viral