Generic Detection of Coronaviruses and Differentiation at the Prototype Strain Level by Reverse Transcription-PCR and Nonfluorescent Low-Density Microarray

Abstract

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

Nucleic acid amplification-based diagnosis of respiratory virus infections

“…The assay reportedly had 1-2 log better sensitivity than the primers recommended by the WHO. Wei et al described a multiplex RT-PCR amplifying HA, N1 and NP genes HCoV OC43 5-30 5 4 [73,177,178,197,202] HCoV 229E 1-5 3 3 [73,179,180,198,202,205] HCoV NL63 1-9 12 9 [73,186,187,190,197,[200][201][202][203] HCoV HKU1 1-11 9 8 [73,192,193,196,197,[202][203][204] the sensitivity of NASBA was equivalent to egg culture for the detection of H5N1 in 94 avian blood and anal swabs [33]. One of the NASBA primers targeted the polybasic amino acid sequence at the cleavage site of the HA0 HA precursor confirming identification of a HPAI.…”

Circulation of Group 2 Coronaviruses in a Bat Species Common to Urban Areas in Western Europe

“…A nested CoV genus-wide reverse transcription (RT)-polymerase chain reaction (PCR) was performed as described previously (de Souza Luna et al 2007). To exclude falsenegative results due to the presence of RT-PCR inhibiting factors, each sample was also analyzed in the presence of a CoV spike RNA at an experimentally predetermined detectable concentration.…”

Highly diversified coronaviruses in neotropical bats

“…RT-PCR covering the subfamily Coronavirinae was done as described previously (de Souza Luna et al, 2007). The 455 bp amplicons from the RNA-dependent RNA polymerase (RdRp) generated by the screening RT-PCR (404 nt after exclusion of PCR primers) were extended towards the 59-end of the genome using virus-specific reverse primers and upstream consensus forward primers, as described previously (Drexler et al, 2010).…”