Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E

Abstract

The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction mixture (5 microl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 10(3) to 10(10) viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 x 10(9) per reaction mixture can be detected, which corresponds to 10(4) to 10(11) viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.

FIG. 1.

Tenfold serial dilutions ranging from 10² to 10⁷ copies of HCoV OC43 cRNA (A) and ranging from 10³ to 10⁸ copies of HCoV 229E cRNA (B) were tested in duplicate in the real-time qRT-PCR. A standard curve graph is made by plotting the C_T values on the y axis and the log of the input amounts on the x axis. The slope of the HCoV OC43 standard curve (A) is −3.54, and the correlation coefficient is 0.999. The HCoV 229E standard curve (B) displays a slope of −3.5 and a correlation coefficient of 1.000.

FIG. 2.

The dynamic range of the assay was tested by running 10-fold serial dilutions of the cRNA standard ranging from 2 to 2 × 10¹² molecules in the reaction. The amplification plot shows the ΔRn on the y axis (where Rn is the fluorescence emission intensity of the reporter dye normalized to a passive reference and ΔRn is the Rn of an unreacted sample minus the Rn of the reaction) against the cycle number displayed on the x axis. (A) The dynamic range of the HCoV OC43 assay spans 7 log units, ranging from 20 to 2 × 10⁸ molecules of HCoV OC43 per reaction mixture, with the corresponding C_T values ranging from 38.88 ± 0.83 for 20 copies to 15.98 ± 0.02 for 2 × 10⁸ copies. (B) For the HCoV 229E assay, a 7-log-unit dynamic range with C_T values ranging from 38.09 ± 1.13 for 2 × 10² copies per reaction mixture to 15.05 ± 0.01 for 2 × 10⁹ copies per reaction mixture is demonstrated.