A chip-based rapid genotyping assay to discriminate between rhinovirus species A, B and C
Section snippets
Background
Human rhinoviruses (RVs) are the most common causative agents of upper respiratory tract infection in humans [1]. They were previously considered to cause only relatively mild illness; yet cumulative associations with severe disease of the respiratory tract, including bronchitis, pneumonia, and exacerbations of chronic pulmonary disease have been reported [[2], [3], [4], [5]]. Additionally, RV infection has been described to induce wheezing in early infancy and is suggested to be an important
Objectives
In this manuscript we describe the development and validation of a novel tool to discriminate between rhinovirus species, using an array of oligonucleotide probes. The Chipron LCD RV array will allow fast detection of a wide spectrum of RV genotypes with good diagnostic sensitivity, including possible dual infections.
Assay and probe design
The developed assay encompasses a single PCR, which includes amplification of the VP4/VP2 region of RV, followed by hybridization of the PCR product on a macro array chip spotted with probes. Each probe covers a number of RV-A, B or C, based on sequence identity within the three different species.
Probes were designed after alignment of the VP4/VP2 region using a large set of RV prototypes (http://www.picornaviridae.com, Genbank, accessed June 2014) (Supplemental Table 1) and secondly the RV
Testing of pre-typed samples
To test whether the Chipron LCD RV array correctly classifies a representative set of RV genotypes, 134 previously typed RV-positive samples including 64 samples containing 37 different RV-A species, 23 samples containing 15 different RV-B species, and 47 samples containing 23 different RV-C species, were tested. In 130/134 samples (97%) covering 74/75 genotypes (98.6%) the species were correctly identified. Only one sample tested RV-C instead RV-A and three samples were tested negative.
Discussion
This study presents the design and evaluation of a RV typing array, discriminating species A, B and C. The RV array has demonstrated to identify 98.6% of a panel of 75 different genotypes in sequenced samples. In previously tested RV positive samples 92.5% were typed correctly, which is comparable to the percentage obtained by sequencing. The Chipron LCD RV array is faster and less laborious than sequencing and allows the detection of multiple genotype infections. To the best of our knowledge,
Funding
Seventh Framework Programme of the European Union AIPP under contract PIAPP-GA-2013-612308.
Conflict of interests
None declared.
Ethical approval
Not required.
Acknowledgements
We thank Rene Minnaar and Sjoerd Rebers of the Laboratory of Clinical Virology (AMC) for technical assistance, Richard Molenkamp of the Laboratory of Clinical Virology (AMC) for helpful discussions and scientific support, and Volker Heiser from Chipron for assisantance in the selection of probes. This work was supported by the Seventh Framework Programme of the European Union AIPP under contract PIAPP-GA-2013-612308 (AIROPico network, www.airopico.eu)
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Cited by (2)
Rhinovirus structure, replication, and classification
2019, Rhinovirus Infections: Rethinking the Impact on Human Health and DiseaseProgress in human picornavirus research: New findings from the AIROPico consortium
2019, Antiviral ResearchCitation Excerpt :A Chipron array utilizing several sets of type-specific probes to detect and distinguish between RV-A, RV-B and RV-C, reduces the time to results considerably. The developed assays were validated in a clinical setting (Westerhuis et al., 2018). In parallel, we developed a low cost workflow for entero- and rhinoviral typing using RT-qPCR, which should expedite the identification of positive samples after PCR amplification and subsequent Sanger sequencing.
- 1
Both authors contributed equally to the manuscript.