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

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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. Sample Population—Urine and blood samples from 6 specific-pathogen-free cats. Procedures—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 1,950, 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 and Clinical Relevance—Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RTPCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCVinduced urinary tract diseases in cats. (Am J Vet Res 2005;66:915–920)