Screening was performed by amplifying a 185 nt fragment in the RdRp region of the ORF1 gene of caliciviruses. Conserved degenerate primers (59-GAYTAYTCNMRRTGGGAYTC-39 and 59-GGCATNCCNGAKGGNAYNCC -39) were designed from the multiple sequence alignment of the available calicivirus gene sequences in NCBI GenBank. First-strand cDNA synthesis was performed using SuperScript III kit (Invitrogen) according to manufacturer's instructions. The PCR mixture (25 ml) contained cDNA, 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 precau-tions were taken to avoid PCR contamination and no false-positive signal was observed in the negative controls. The PCR products were gel-purified using a QIAquick gel extraction kit (Qiagen). Both strands of the PCR products were sequenced twice with an ABI Prism 3730xl DNA Analyser (Applied Biosystems), using the two PCR primers.