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

Figures

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  Figure 1. A schematic summary of the RT-C2CA assay representing the key steps (I). Egg inoculation via allantoic fluid route and virus RNA isolation (II). Reverse transcription (RT) using biotinylated primers (III). Hybridization of padlock probe (PLP) to the target, followed by ligation (IV). Capture of ligated products on magnetic beads; (V). Rolling circle amplification (RCA) of captured molecules (VI). Monomerization of RCA products (RCPs) (VII). Ligation of monomerized fragments and second RCA (VIII). Fluorescence labelling of RCPs, followed by digital quantification using amplified single molecule detection (ASMD) – the inset shows an output image from the instrument, where individual RCPs become visible as single bright dots (in white, compared to the dark background) during analysis. Arrow indicates the polarity of the sequence from 5′ end to 3′ end.

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  Figure 2. A schematic representation of PLP design strategy: (A). Multiple sequence alignment (MSA) of 335 NDV L-gene sequences using Geneious version 6.1.8, where the disagreements to the consensus sequences are highlighted in different colors; (B). Construction of unrooted phylogenetic tree using Neighbour-Joining method, showing the two groups A and B and the respective haplotypes; (C). Location of chosen target sites for PLP design, along the length of L-gene; (D). Extracted regions of L-gene sequences from the alignment view, representing the scoring for mismatches in the individual sequences relative to the consensus; seen below is the design of mutation-tolerant PLPs; seen on the right is the list of generated PLPs showing the inclusion of Wobbles.

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  Figure 3. (A) Optimization of RT parameters for the choice of primers (RP: Random Primers, SSP: Single Specific Primers, SP mix: mixture of individual specific primers) for Herts/33 and HU-14036/06; 1:10 dilution of cDNA synthesized using SSP B1 and B3, respectively, and amplified using PLP B1 and B3 were used. (B) Performance of individual and cocktail PLPs for C2CA validation using two chosen strains, along with their respective negative controls (water instead of template); cDNA dilution of 1:20, synthesized using RP.

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  Figure 4. Heat map showing the detection of NDV isolates in two experimental batches obtained from two different sources. The specificity controls with other related viruses are included alongside. For Batch-I, 1:50 dilution of cDNA, and for Batch-II, a final copy number of 104 was used for the samples. The RCP counts obtained by C2CA are illustrated as intensity difference in the heat map (n = 2). X symbol denotes no performance of experiments with these conditions.

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  Figure 5. Estimation of limit of detection (LoD) for two chosen strains using C2CA assay and ASMD read-out. LoD was estimated from the linear fit (obtained from the log values) and the background (calculated as 257 RCPs, the mean negative control (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{{\rm{x}}}$$\end{document}x¯) + 3 st. dev. (σ) from the mean), represented in the figure.

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  Figure 6. Multiplexing of hypervariable RNA avian viruses using microfluidic membrane enrichment. (A) A table showing the RCP values obtained from membrane enrichment readout (n = 2) for the detection of isolated RNA from field strains using respective PLP cocktail for specificity testing with their matching and non-matching targets, along with water negative control. Inset shows images corresponding to the performance of NDV PLPs on various individual targets. Scale bar = 20 μm. (B) The multiplexing performance of cocktail PLPs to detect the mixture of three chosen targets is represented using only NDV-specific cocktail PLPs as well as complete cocktail set; specific fluorophores tagged for each of the viruses were used: NDV – Cy3 (green); IBV – Cy5 (red); AIV – Cy7 (blue). Inset shows merged 3-channel images corresponding to each condition. Error bars are derived from triplicate measurements of membrane-enriched samples for complete cocktail, and duplicate measurements for NDV cocktail experiments.