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

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

doi:10.1038/s41598-019-39854-3

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.

Highlights

The ever-increasing numbers of viruses are posing a great threat by causing unexpected health issues, including epidemics, in both humans and animals
The current study includes a strategic design for molecular detection of rapidly evolving virus strains using Padlock probes (PLPs) and C2CA with Newcastle Disease Virus (NDV) as a model
The biggest challenge in disease control of single-stranded RNA viruses is due to their high mutation rate that leads to ever evolving new strains and possible epidemics

Summary

Introduction

The ever-increasing numbers of viruses are posing a great threat by causing unexpected health issues, including epidemics, in both humans and animals. In order to combat the issue of handling such emerging strains, a WHO/OIE panel has called for timely and responsive disease surveillance systems for both, animal and human populations[4], throwing light on currently available poor information related to viral subpopulations or quasispecies, which in turn hinders their accurate diagnosis This problem is prominent among poultry viruses, where a high number of strains pose a serious threat to both, meat and egg production, as well as risk of turning into human epidemics. The most widely used techniques for detection and typing of NDV strains include isolation of virus and immunohistochemistry, as well as RT-PCR, gene sequencing and microarrays[14,15] All these methods have strengths and weaknesses in terms of sensitivity and specificity, assay integration, automation and field-based diagnosis. We integrated a mutation-tolerant PLP design and the membrane-based detection tool to demonstrate a sensitive PLP-based C2CA assay for the detection of highly mutating viral strains

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