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.
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.
Scansen
Brian A
BA
Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
Kruger
John M
JM
Wise
Annabel G
AG
Venta
Patrick J
PJ
Bartlett
Paul C
PC
Maes
Roger K
RK
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
D012367:RNA, Viral