Advances in the differential molecular diagnosis of vesicular disease pathogens in swine

Figures

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  Figure 1. Schematic illustration of ddPCR. This figure illustrates the digital PCR droplet principle and how a single sample containing the target sequence is partitioned. PCR amplified produces thousands of copies that can be detected and interpreted by a detection system. In a typical ddPCR workflow, a single sample includes target and nonspecific sequences (DNA or RNA), real-time PCR primers and fluorescent-labeled probes, and standard real-time PCR master mixes. (1) A sample partitioned into thousands of single nanoliter droplets with the generation of water-in-oil emulsions. A proportion of droplets contain no template molecules, while others contain one or more targets. The generation of droplets from the sample is achieved in several ways. Standard methods include T-junction and flow-focusing geometry. Only the flow-focusing geometry is shown here. For more details, see the text. (2) Traditional end-point PCR is then performed to amplify the target sequence. (3) Target sequence droplets exhibit higher fluorescence intensity and are known to be positive droplets. Empty or no targets show low and negative fluorescent intensity. This fluorescence intensity versus time is plotted on a…

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  Figure 2. Amplification via loop-mediated isothermal amplification (LAMP) and various detection methods. (A) Diagram of LAMP primers located on the target sequence and the amplification process. The FIP primer binds to its complementary sequence and begins amplifying the first strand, followed by binding and amplification of the second strand by the F3 primer. The following steps involve the amplification of the FIP-amplified strand by the B3 primer. Finally, FIP and BIP continue the LAMP cycle, resulting in dumbbell-shaped amplicons. A full stepwise description of the individual steps is provided in the figure. (B) Magnesium pyrophosphate-based visual detection of the LAMP product. During LAMP, a large amount of pyrophosphate ion is produced as a by-product, which reacts with magnesium provided in the reaction mixture. The resulting product, magnesium pyrophosphate, forms a white precipitate that allows easy visual detection. The turbidity of the final LAMP reaction confirms the presence or absence of the targeted nucleic acid. (C) Calcein-based fluorescence detection of the LAMP product. The reaction of the by-product pyrophosphate with magnesium or calcein-manganese aids in the…

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  Figure 3. Recombinase polymerase amplification (RPA)-based amplification and its detection methods. (A) Schematic diagram of the RPA assay. An ideal RPA assay consists of forward and reverse primers, a recombinase protein that helps bind primers to the target nucleic acid, ssDNA-binding proteins to stabilize the ssDNA, and a polymerase to amplify the primer-bound strands. The stepwise amplification process is shown in the figure. (B) Exonuclease-based detection of the RPA product. A 46–52 bp long Exo probe flanked by a quencher and fluorophore binds to the amplified product. Exo probe contains a THF (tetrahydrofuran) residue known as dSpacer, which is cleaved by the exonuclease, thus releasing the fluorophore from the quencher. The observed fluorescent signal indicates the presence of the target nucleic acid. (C) Endonuclease-based detection of the RPA product. A 46–52 bp long oligonucleotide probe labeled with FAM or Alexa fluor binds to the target strand. The annealed probe is cleaved by the nfo enzyme, freeing the 3′-OH group of the probe and is used as a primer in the subsequent reactions. The resulting amplicons are produced with FAM and biotin using a biotin-labeled reverse primer.…

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  Figure 4. Schematic of the SHERLOCK and DETECTR. (A) CRISPR-Cas13a based detection (SHERLOCK). Following the RPA (Recombinase Polymerase Amplification) or RT (Reverse Transcription)-RPA, the RNA target recognized by Cas13a-crRNA complex is produced by in vitro transcription of the DNA amplicons. On recognition of the RNA target, the trans-cleavage activity of Cas13a is activated, resulting in the degradation of the doubly labeled fluorescent reporter. (B) CRISPR-Cas12a based detection (DETECTR). The RPA amplicon is used directly as the target for the Cas12a-crRNA complex. On the recognition of the DNA target, the trans-cleavage activity of Cas12a is activated, resulting in the degradation of the doubly labeled fluorescent reporter (Yin et al., 2021).