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Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E.
J Clin Microbiol. 2005 Nov; 43(11):5452-6.JC

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.

Authors+Show Affiliations

Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16272469

Citation

Vijgen, Leen, et al. "Development of One-step, Real-time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E." Journal of Clinical Microbiology, vol. 43, no. 11, 2005, pp. 5452-6.
Vijgen L, Keyaerts E, Moës E, et al. Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E. J Clin Microbiol. 2005;43(11):5452-6.
Vijgen, L., Keyaerts, E., Moës, E., Maes, P., Duson, G., & Van Ranst, M. (2005). Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E. Journal of Clinical Microbiology, 43(11), 5452-6.
Vijgen L, et al. Development of One-step, Real-time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E. J Clin Microbiol. 2005;43(11):5452-6. PubMed PMID: 16272469.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E. AU - Vijgen,Leen, AU - Keyaerts,Els, AU - Moës,Elien, AU - Maes,Piet, AU - Duson,Griet, AU - Van Ranst,Marc, PY - 2005/11/8/pubmed PY - 2005/12/31/medline PY - 2005/11/8/entrez SP - 5452 EP - 6 JF - Journal of clinical microbiology JO - J Clin Microbiol VL - 43 IS - 11 N2 - 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. SN - 0095-1137 UR - https://www.unboundmedicine.com/medline/citation/16272469/Development_of_one_step_real_time_quantitative_reverse_transcriptase_PCR_assays_for_absolute_quantitation_of_human_coronaviruses_OC43_and_229E_ L2 - http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=16272469 DB - PRIME DP - Unbound Medicine ER -