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

Scientific Reports
Sibel CiftciMats Nilsson

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 ...Continue Reading

References

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Citations

Jul 2, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Siraprapa BoobphahomKwanwoo Shin
Jun 26, 2020·Veterinary Sciences·Matteo LegnardiMattia Cecchinato
Aug 28, 2020·Reviews in Medical Virology·Arefeh BasiriNima Rezaei
Jun 3, 2021·Biosensors·Darío Sánchez MartínTeresa Zardán Gómez de la Torre
Oct 17, 2021·Molecular Biotechnology·Mehrnaz EntesariFariba Rafiei

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Methods Mentioned

BETA
deamination
nucleic acid amplification
chip
PCR

Software Mentioned

J ava Al ignment View er
BLASTn
CLC Genomics Workbench (
CLC Genomics Workbench
Geneious Tree builder
Jalview (
CellProfiler
Geneious
Zen

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