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Molecular detection of human rhinoviruses in respiratory samples: a comparison of Taqman probe-, SYBR green I- and BOXTO-based real-time PCR assays

Julien Dupouey12*, Laetitia Ninove12, Vanessa Ferrier1, Odile Py1, Céline Gazin2, Laurence Thirion-Perrier1 and Xavier de Lamballerie12

Author Affiliations

1 IRD French Institute of Research for Development, EHESP French School of Public Health, Aix Marseille Univ, UMR_D 190 “Emergence des Pathologies Virales”, Marseille 13005, France

2 Pôle des Maladies Infectieuses et tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, 264 rue Saint-Pierre 13385, Marseille CEDEX 05, France

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Virology Journal 2014, 11:31  doi:10.1186/1743-422X-11-31

Published: 18 February 2014

Abstract

Background

Human Rhinoviruses (HRV) are major causative agents of acute respiratory tract infections in all age group and important contributing factors of childhood morbidity and mortality. Clinical presentation is poorly specific and the great antigenic and genetic variability of HRVs renders the biological diagnosis complex. Here, we have evaluated several molecular diagnostic protocols, including Taqman probe-based and intercalating agent-based RT-PCR assays.

Methods

5,627 respiratory samples sent to the laboratory of Virology of the University Hospitals of Marseille, France, from March 2011 to February 2012, were tested using a real-time RT-PCR assay in the 5’NCR of the rhinoviral genome that associated a Taqman probe and the detection of DNA-BOXTO-dye complexes. A sample of 500 BOXTO-positive samples were further tested using the same probe assay (without BOXTO), and a SYBR Green assay (using the same amplification primers). The specific amplification of HRV sequences was assessed by NGS amplicon sequencing.

Results

The Taqman probe RT-PCR assay identified 696/5,627 samples (12,4%) as HRV-positive. BOXTO-positive samples included all probe-positive samples and 1,913 additional samples, of which only 24.3% were confirmed by sequencing. The SYBR Green assay was more specific (16/550 samples were probe-negative/SYBR Green-positive, all confirmed by 5′NCR sequencing), but 3/500 samples were probe-positive/SYBR Green-negative.

Conclusions

Our results highlight the difficulty in detecting HRVs in clinical samples using a single molecular detection system. Amongst the 3 systems tested, the best compromise was obtained with the SYBR Green assay, which, by comparison with our probe-based assay provided an improved sensitivity without altering the detection specificity. Interestingly, a majority of probe-negative/BOXTO- or SYBR Green-positive samples were not associated with mutations in the sequence targeted by the probe. Sequence-based modifications of the secondary structure of the HRV 5′NCR may be associated with a limited access to the probe hybridisation region. Further investigations may identify a test combining a probe based- and an intercalating agent-based detection, which will significantly improve the diagnosis of HRV infections.

Keywords:
BOXTO; SYBR green; Taqman probe; Picornaviridae; Human rhinovirus; Respiratory infections; Molecular diagnosis