A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.Coronaviruses (CoV) (family Coronaviridae, order Nidovirales) are large enveloped RNA viruses with a 27-to 32-kb genome of positive polarity. They comprise a very diverse spectrum of pathogens of humans and animals (2, 7). The coronavirus etiology of severe acute respiratory syndrome (SARS) and the recent discoveries of the novel human coronaviruses (hCoV) NL63 and HKU1 (5, 13, 15) have triggered intensified efforts in virus identification and diagnostics. Generic reverse transcription (RT)-PCR assays with a very broad detection range are required, but few such assays are available. None of them has been previously validated in a diagnostic setting (9, 12).The requirement for sequencing in order to achieve strain identification limits the applicability of generic PCR assays in general. Alternative techniques, such as mass spectrometry or complex fluorescent DNA microarrays, have been proposed (10), but these will often be too sophisticated for medical facilities. We describe here a simple and feasible approach to detecting the full spectrum of coronaviruses with diagnostic sensitivity, combining generic RT-PCR and low-cost, low-density (LCD) DNA microarrays which can be read with the naked eye.Primers for universal RT-PCR for the genus Coronavirus were designed after aligning all coronavirus RNA-dependent RNA polymerase genes. RNA-dependent RNA polymerase motifs A and C were targeted because they contain short amino acid patterns that are 100% identical in all coronaviruses (16). Primer binding regions corresponded to patterns LMGWDYPKCD and MMILSDDAV, comprising domains essential for metal ion chelation and binding of the primer 3Ј-end/template complex (11, 16). Reactions (25-l mixtures) were carried out using the QIAGEN (Hilden, Germany) one-step RT-PCR kit, with 200 nM of primer PC2S2 (equimolar mixture of TTATGGGTTGGGAT TATC and TGATGGGATGGGACTATC), 900 nM of primer PC2As1 (equimolar mixture of TCATCACTCAGAATCATCA, TCATCAGAAAGAATCATCA, and TCGTCGGACAAGATC ATCA), 1 l QIAGEN one-step RT-PCR kit enzyme mix, and 5 l RNA extract. The amplification procedure comprised 30 min at 50°C; 15 min at 95°C; 10 cycles of 20 s at 94°C, 30 s starting at 62°C with a decrease of 1°C per cycle, and 40 s at 72°C; and 30 cycles of 20 s at 95°C, 30 s at 52°C, and 40 s at 72°C. To determine the sensitivity of the assay, the target regions including sufficient stretches of flanking sequence were cloned from several coronaviruses (Table 1) and transcribed into RNA (3, 4). Amplification