DNA was extracted from all samples using QIAamp DNA Mini kit (Qiagen) according to manufacturer's instructions, and then subjected to PCR using 2 different sets of screening PCR primers. The primer sequences are: forward primer A 59-CCCGCKAS-TACWGGNAARAC -39 and reverse primer A 59-CCGTAAYT-CKRCCYTCKTCCCA -39 (targeting a 148 bp fragment); forward primer B 59-TCTGCTATTGTAATHAARGAYGT-39 and reverse primer B 59-AAACACTCTGCRTCRTGRTGYTC-39 (targeting a 293 bp fragment). The primers were designed from multiple alignments of the nucleotide sequences of VP2 regions of PARV4 and related parvoviruses using our previously described strategy [19] . The PCR mixture (25 ml) contained DNA extracted from samples, PCR buffer (10 mM Tris/HCl pH 8.3, 50 mM KCl, 2 mM MgCl 2 and 0.01% gelatin), 200 mM of each dNTP and 1.0 U AmpliTaq Gold polymerase (Applied Biosystems). PCR cycling conditions were as follows: hot start at 94uC for 7 min, followed by 50 cycles of 94uC for 1 min, 50uC for 1 min and 72uC for 1 min with a final extension at 72uC for 10 min in an automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid PCR contamination and no false positive results was observed in negative controls.