TABLE 1.

PCR primers and probes used in the multiplex real-time PCR assay

Primer or probea	Primer or probe sequence (5′→3′)	Region of gene (nt)b
28S rDNA
Forward primer	CGCTAATAGGGAATGTGAGCTAGG	663-686
Reverse primer	TGTCTGAACCTCCAGTTTCTCTGG	783-760
Molecular beacon	Texas red-CGCGCACCCTACTGATGATGTGTTGTTGCCGCGCG-DABCYL	716-740
C. felis OmpA
Forward primer	GAACTGCAAGCAACACCACTG	281-301
Reverse primer	CCATTCGGCATCTTGAAGATG	357-337
FQ probe	6-FAM-CGCTGCCGACAGATCAAATTTTGCC-BHQ1	303-327
FHV TKc
Forward primer	GGACAGCATAAAAGCGATTG	173-192
Reverse primer	AACGTGAACAACGACGCAG	247-229
FQ probe	Cy5-AATTCCAGCCCGGAGCCTCAAT-BHQ2	201-222

^aAll primers and probes were synthesized by Cruachem Ltd., Glasgow, Scotland. The 28S rDNA molecular beacon was labeled with Texas red at the 5′ end and a 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) quencher at the 3′ end. The regions of stem-loop formation are underlined. FQ probes were labeled with either 6-carboxyfluorescein (6-FAM) or cyanine 5 (Cy5) at the 5′ end and the appropriate black hole quencher (BHQ) at the 3′ end.

^bThe following gene sequences were used: feline 28S rDNA (GenBank accession number AF353617), C. felis major outer membrane protein gene (GenBank accession number AF269258) and FHV thymidine kinase gene (GenBank accession number M26660). nt, nucleotides.

^cTK, thymidine kinase.