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

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.

Microfluidic devices for detection of RNA viruses

“…In another study, Ciftci et al showed that traditional approaches like virus isolation, serology, immunoassays, and RT‐PCR are difficult and limited in terms of specificity and sensitivity for detecting RNA viruses. However, rolling circle amplification, in combination with padlock probes, had a higher specificity for detecting RNA viruses like Newcastle disease virus, avian coronavirus, and avian influenza virus 78 …”

Infectious Bronchitis Virus Evolution, Diagnosis and Control

“…Other molecular techniques, such as loop-mediated isothermal amplification (LAMP) and padlock probes (PLPs) combined with rolling circle amplification (RCA), have been validated for IBV detection [ 78 , 79 ]. Additionally, NGS techniques have recently been used to study IBV [ 80 , 81 ].…”

Recent Advances in Microfluidic Paper-Based Analytical Devices toward High-Throughput Screening

“…These techniques include, but are not limited to, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), helicase-dependent amplification (HDA), rolling-circle amplification (RCA), signal-mediated amplification of RNA technology (SMART), strand-displacement amplification (SDA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), which is probably the most well-known of these techniques [ 163 , 164 ]. Generally, the above-mentioned techniques target specific and unique sequences of DNA [ 22 , 165 ] or RNA [ 166 , 167 ] and in many cases, microRNA (miRNA) [ 168 , 169 , 170 ]. Paper-based nucleic acid amplification and detection are useful in diseases diagnosis [ 22 , 23 ], forensic analysis [ 171 ], food [ 172 ] and beverage [ 173 ] control, and environmental testing [ 165 ].…”