title: In vitro comparison of RNA preparation methods for detection of feline calicivirus in urine of cats by use of a reverse transcriptase-polymerase chain reaction assay.
abstract: OBJECTIVE
  To compare 5 methods of preparation of RNA from feline urine samples for use in a feline calicivirus (FCV), p30 gene-based, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay.
  
  
  METHODS
  Urine and blood samples from 6 specific-pathogen-free cats.
  
  
  METHODS
  Aliquots of each urine sample (unmodified, centrifuged, or mixed with whole or hemolyzed blood) were spiked with FCV and serially diluted in urine. Serial dilutions of FCV in tissue culture medium were used as positive controls. Viral RNA was prepared via dilution and thermal inactivation (DT method), polyethylene glycol precipitation (PEG method), isolation with oligo(dT)25-coated magnetic beads (dTMB method), or extraction by use of 2 silica gel-based columns (RN or QA method). Lower detection limits and mean RT-PCR threshold cycle (Ct) values associated with each RNA preparation method and sample type were compared.
  
  
  RESULTS
  Because DT-prepared samples yielded negative results via RT-PCR assay, this method was not evaluated. Lower detection limits (TCID50/sample) for the assay in urine were 1950, 104, 11, and 7 for PEG-, dTMB-, RN-, and QA-prepared samples, respectively. For RN and QA preparations, Ct values were similar and significantly lower than those for dTMB and PEG preparations. Overall, urine modifications did not affect FCV RNA detection in dTMB-, QA-, and RN-prepared samples.
  
  
  CONCLUSIONS
  Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RT-PCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCV-induced urinary tract diseases in cats.
authors.affiliation: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Scansen
authors.forename: John M
authors.initials: JM
authors.lastname: Kruger
authors.forename: Annabel G
authors.initials: AG
authors.lastname: Wise
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Venta
authors.forename: Paul C
authors.initials: PC
authors.lastname: Bartlett
authors.forename: Roger K
authors.initials: RK
authors.lastname: Maes
mesh_terms: D000818:Animals; D017250:Caliciviridae Infections / Q000097:blood / Q000175:diagnosis / Q000652:urine / Q000662:veterinary*; D017927:Calicivirus, Feline / Q000235:genetics / Q000302:isolation & purification*; D002371:Cat Diseases / Q000175:diagnosis* / Q000821:virology; D002415:Cats; D005260:Female; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification* / Q000652:urine*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013047:Specific Pathogen-Free Organisms
chemical_list: D012367:RNA, Viral