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

• Published in: Virology journal Vol. 11; no. 1; p. 31
• Main Authors: Dupouey, Julien, Ninove, Laetitia, Ferrier, Vanessa, Py, Odile, Gazin, Céline, Thirion-Perrier, Laurence, de Lamballerie, Xavier
• Format: Journal Article
• Language: English
• Published: England Springer-Verlag 18.02.2014; BioMed Central Ltd; BioMed Central
• Subjects: Adolescent; Adult; Age; Aged; Aged, 80 and over; assays; Child; Child, Preschool; childhood; Comparative analysis; Diagnosis; Ear diseases; Enterovirus; Epidemiology; Experiments; France - epidemiology; Genetic aspects; Genetic diversity; genetic variation; genome; Genomes; Health aspects; Hospitals; Human health and pathology; Human rhinovirus; Humans; hybridization; Infant; Infant, Newborn; Infections; Infectious diseases; Investigations; Life Sciences; Male; Methods; Microbiology and Parasitology; Middle Aged; Molecular Diagnostic Techniques - methods; morbidity; Mortality; mutation; Physiological aspects; Public health; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; respiratory tract diseases; Respiratory Tract Infections - diagnosis; Respiratory Tract Infections - epidemiology; Respiratory Tract Infections - virology; reverse transcriptase polymerase chain reaction; Rhinovirus - classification; Rhinovirus - genetics; Rhinovirus - isolation & purification; Rhinoviruses; sampling; Sequence Analysis, DNA; Virology; Young Adult; France; BOXTO; Molecular diagnosis; Picornaviridae; Respiratory infections; SYBR green; Taqman probe; Human rhinovirus
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/1743-422x-11-31

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.