A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses

Sibel Ciftci, Felix Neumann, Iván Hernández-Neuta, Mikhayil Hakhverdyan, A. Bálint, David Herthnek, Narayanan Madaboosi, Mats Nilsson

Research output: Contribution to journalArticle

Abstract

The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.

Original languageEnglish
Article number2872
JournalScientific reports
Volume9
Issue number1
DOIs
Publication statusPublished - Dec 1 2019

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Newcastle disease virus
RNA Viruses
Mutation
Viruses
Genotype
Sensitivity and Specificity
Microfluidics
Molecular Pathology
Sequence Alignment
Viral RNA
Virus Diseases
Mutation Rate
Serology
Poultry
Immunoassay
Nucleic Acids
Limit of Detection
Genome
Polymerase Chain Reaction

ASJC Scopus subject areas

  • General

Cite this

Ciftci, S., Neumann, F., Hernández-Neuta, I., Hakhverdyan, M., Bálint, A., Herthnek, D., ... Nilsson, M. (2019). A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses. Scientific reports, 9(1), [2872]. https://doi.org/10.1038/s41598-019-39854-3

A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses. / Ciftci, Sibel; Neumann, Felix; Hernández-Neuta, Iván; Hakhverdyan, Mikhayil; Bálint, A.; Herthnek, David; Madaboosi, Narayanan; Nilsson, Mats.

In: Scientific reports, Vol. 9, No. 1, 2872, 01.12.2019.

Research output: Contribution to journalArticle

Ciftci, S, Neumann, F, Hernández-Neuta, I, Hakhverdyan, M, Bálint, A, Herthnek, D, Madaboosi, N & Nilsson, M 2019, 'A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses', Scientific reports, vol. 9, no. 1, 2872. https://doi.org/10.1038/s41598-019-39854-3
Ciftci, Sibel ; Neumann, Felix ; Hernández-Neuta, Iván ; Hakhverdyan, Mikhayil ; Bálint, A. ; Herthnek, David ; Madaboosi, Narayanan ; Nilsson, Mats. / A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses. In: Scientific reports. 2019 ; Vol. 9, No. 1.
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