Twenty µL of the PCR reaction underwent electrophoresis on a 1% low melting point agarose gel. DNA bands (365-bp product) were excised from the gel and purified by using Wizard PCR Preps DNA Purification System (Promega Corp., Madison, WI, USA). The PCR product was ligated into the pCR 2.1-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, CA, USA.). An SFV DNA plasmid (pSFV-1Lgp), representing long terminal repeat, gag, and pol of SFV-1, was included as a positive PCR control and for determining sensitivity of detection by serial dilution (provided by A. Mergia). TOP10 cells were transformed with the ligation reaction, plated onto Luria broth agar plates containing 50 µg/mL kanamycin, and incubated overnight at 37°C. Miniscreen DNA was purified by using Wizard Plus Minipreps DNA Purification System (Promega). Samples were sequenced with the ABI 373 automated fluorescent sequencer using BigDye Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA, USA).