In an attempt to improve the sensitivity of HKU1 detection, we analyzed samples with negative results by PCR with a nested PCR specifi c for coronavirus HKU1. This nested assay was designed on an alignment of our HKU1-positive sample sequence (BRA169) and different HKU1 genotype sequences deposited in GenBank. Primers Fn-HKU1 (forward 5′-CGTGCYA TGCCAAATATTTTGCG-3′, HKU1-NC_006577, nt 15433-15454) and Rn-HKU1 (reverse 5′-TAGCAACC GCCACACATAAC-3′, HKU1-NC_ 006577, nt 15562-15581) produced an amplicon of 149 bp. The nested PCR was run in a 50-μL reaction comprising 10 μL of PCR product, 1.5 units of DNA Polymerase (Biotools, Madrid, Spain), 1 μmol/L of each primer, 200 μmol/L of each dNTP (Applied Biosystems), 2 mmol/L MgCl 2 , and 1× buffer. PCR mixtures were heated to 95°C for 5 min, followed by 35 cycles of 1 min at 95°C, 30 s at 62°C, and 40 s at 72°C, followed by a fi nal 10 min at 72°C. Clinical samples positive for HKU1 were used as positive controls in the HKU1 nested PCR. All fragments obtained from PCR and nested PCR were analyzed in a 2% (wt/ vol) agarose gel by electrophoresis, stained with 0.5 μg/mL of ethidium bromide, and subsequently sequenced to confi rm the type of coronavirus. Nucleotide sequencing reactions were performed on both amplicon strands by using an ABI PRISM Big Dye Cycle Sequencing Kit with the ABI PRISM 3100 automatic sequencer (Applied Biosystems).