Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction

Abstract

An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID50 of HCoV-229E and 0.01 TCID50 of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.

Fig. 1

Ethidium bromide staining of a 2% agarose gel showing tenfold dilutions of a HcoV-OC43 suspension positive or negative for RT-PCR HcoV-OC43, gene M (334 pb) and heminested-RT-PCR HcoV-OC43, gene M (169 pb), and the correspondent hybridization index. Lane 1 and 9, molecular weight marker (100 pb); lane 2: pure viral suspension RT-PCR; lane 3, dilution 10⁻¹ RT-PCR; lane 4, dilution 10⁻² RT-PCR; lane 5, dilution 10⁻³ RT-PCR; lane 6, dilution 10⁻⁴ RT-PCR; lane 7, RT-PCR positive control; lane 8, RT-PCR negative control; lane 10, pure viral suspension 1/2 nested RT-PCR; lane 11, dilution 10⁻¹ 1/2 nested-RT-PCR; lane 12, dilution 10⁻² 1/2 nested RT-PCR; lane 13, dilution 10⁻³ 1/2 nested-RT-PCR; lane 14, dilution 10⁻⁴ 1/2 nested RT-PCR; lane 15, 1/2 nested RT-PCR positive control; lane 16, 1/2 nested-RT-PCR negative control.