Development of One-Step, Real-Time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E

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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 × 108 RNA copies per reaction mixture (5 μl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 103 to 1010 viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 × 109 per reaction mixture can be detected, which corresponds to 104 to 1011 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.

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