A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species

Figure 1. Spin-Tube assay: Merging of dynamic chemistry with a simple colorimetric end-point assay in a novel micro Spin-Tube device for analysing “Single Nucleotide Fingerprint” (SNF) markers. Step 1: modification of abasic PNAs, which are immobilized onto nylon membranes following specific spot patterns, with the biotinylated aldehyde-modified cytosine (SMART-C-Biotin). This process requires three steps: (i) perfect hybridization between abasic PNAs and PCR amplicons; (ii) generation of a reversible iminium specie between the secondary amine of the “abasic” unit and the aldehyde group of the SMART-C-Biotin nucleobase driven by the templating nucleobase. In this case, SMART-C-Biotin incorporation can be just templated by a guanidine as otherwise the iminium specie is not stable enough to be reduced; and (iii) reduction of the iminium specie by sodium cyanoborohydride to yield a non-reversible tertiary amine within the PNA printed onto the nylon membrane. Step 2: (iv) Biotin labelling with streptavidin alkaline phosphatase (Streptavidin-ALP); (v) Incubation with the chromogenic substrate (NBT/BCIP, nitro blue tetrazolium chloride/ 5-Bromo-4-chloro-3-indolyl phosphate) which generates a…

Figure 2. (A) Multiple sequences alignment of L. majorvs T. cruzi. Forward (highlighted in yellow) and reverse (highlighted in green) primers. Respectively, highlighted in red and blue, the abasic PNA1 and PNA2 complementary region in the PCR amplicon. Sequence alignment shows 5 SNFs (emphasized with orange rectangles). A red arrow indicates the guanidine “G” nucleotide at +32 from the 5′-terminus under interrogation via abasic PNA1 probe (emphasized also with a red rectangle), while the abasic PNA2 probe interrogates the first SNF (SNF1 emphasized with the blue rectangle). (B) Schemes of region of the PCR amplicons interrogated using the two abasic PNA probes. The chiral PNA monomers are identified with a star and highlighted in green. Incorporation of SMART-C-Biotin provides proof-reading, indicating the presence of the parasitic genome, as well as the discrimination between L. major vs T. cruzi. The white letters within the red and blue squares indicate the templating nucleotides that lie opposite the abasic position of PNA1 and PNA2, represented with the red or blue question mark respectively. (C) Each parasite gives rise to a specific SMART-C-Biotin incorporation pattern. T. cruzi…

Figure 3. (A) Spin-Tube prototype; (B) Plastic components for the Spin-Tube fabrication (Fig. S1 in SI); (C) Amide formation between pre-activated carboxylic acid groups of nylon membranes and primary amine groups of abasic PNAs; (D) Graphic layout of the array: in yellow, 2 spots of biotin markers; in blue, 3 spots of abasic PNA2; in red, 3 spots of abasic PNA1.

Figure 4. Steps of the Spin-Tube assay: (i) Samples collection (minutes) and nucleic acids extraction, either DNA (red) or RNA (green) (5 minutes); (ii) Singleplex PCR amplification of gDNA (90 minutes); (iii) Total RNA fragmentation (15 minutes); (iv) Biotin labelling by dynamic chemistry reaction. The reaction is carried out at 45 °C inside the internal column for the assay of Spin-Tube (60 minutes); (v) Washing with centrifuge (6000 rpm) and SMART-C-Biotin labelling with streptavidin-ALP (5 minutes); (v) Washing with centrifuge (6000 rpm) and addition of colorless chromogenic reagent to form a blue precipitate (8 minutes); (vi–vii) Washing and drying with centrifuge enable an unequivocal identification of the parasite present in the sample. Each parasite gives a unique colorimetric pattern: respectively (a) and (b) spot patterns for L. major and T. cruzi.

Figure 5. (A) Capillary electrophoresis and (B) colorimetric results. First row shows the template used for the PCR reaction (1 to 4). Second and third rows show the capillary electrophoresis results (1A to 4A). Fourth and fifth rows show the colorimetric results (1B to 4B). Regarding columns: Column 1 corresponds to the PCR negative control (water) showing the absence of PCR contamination (1A) and the look of the membrane after all the procedure with just the biotin positive control of the colorimetric assay (1B). Column 2 shows the PCR performed on 20 ng of human gDNA highlighting the specificity of the previously designed primers (2A and 2B). Column 3 matches with the PCR performed on 20 ng of T. cruzi gDNA sample that has been mixed with an equal amount of human gDNA (3A and 3B) and column 4 shows the experiment carried out on 20 ng of L. major gDNA samples mixed with other 20 ng of human gDNA (4A and 4B).

Figure 6. (A) Spotting layout with 3 Biotin marker spots (yellow) rather than the 2 of Fig. 3D. 3 spots of PNA1 (red) and 3 spots of PNA2 (blue). (B) Agilent Bioanalyzer 2100 gel-like images extrapolated from the capillary electrophoresis for the PCR products of T. cruzi. These gel-like images are produced from the chromatogram of the capillary electrophoresis by the bioanalyzer analysis software. NC: negative control PCR (water); 1–6 PCR reactions using decreasing amounts of gDNA of T. cruzi. 1: 50 ng; 2: 5 ng; 3: 0.5 ng; 4: 0.05 ng; 5: 0.005 ng; 6: 0.0005 ng. (C) Decreasing amounts of PCR products were used as templates for the dynamic chemistry reaction to incorporate the SMART-C-Biotin. Positive signals were obtained on both abasic PNA probes with PCR starting concentration from 50 ng up to 0.05 ng (8.7 copies/µL) of template what coincides with the last PCR product that was able to be detected by capillary electrophoresis. A percentage of the relative intensity was calculated using as 100% signal the average of the three biotin marker spots.

Figure 7. Analysis of spiked parasite gDNA in blood. (A) Capillary electrophoresis and (B) Spin-Tube results. First row indicate the template used for the PCR reaction (1 to 4). Second and third rows show the capillary electrophoresis results (1A to 4A). Fourth and fifth rows show the Spin-Tube results (1B to 4B). Regarding columns: Column 1 corresponds to the PCR negative control showing the absence of contamination (1A) and the look of the membrane after all the colorimetric procedure with just the biotin positive control of the colorimetric assay (1B). Column 2 shows the PCR performed on 450 ng of DNA extracted from 200 µL of human blood which highlights the specificity of the previously designed primers and the absence of false negatives due to the presence of human blood and DNA (2A and 2B). Column 3 contains the PCR performed on 450 ng of DNA extracted from 200 µL of human blood spiked-in with 10 ng of L. major gDNA (3A and 3B) and column 4 shows the results of the experiment carried out with a PCR performed on 450 ng of DNA extracted from 200 µL of human blood spiked-in with 0.1 ng of L. major gDNA (4A and 4B).

Figure 8. (A) Scheme for the synthesis of ribosomal RNA (sense RNA). The sense RNA is transcribed from the anti-sense DNA strand by RNA polymerase. The sense RNA strand templates the dynamic chemistry reaction containing both the guanidine “G” and the SNF1 for templating the SMART-C-Biotin incorporation respectively in the abasic PNA1 and PNA2 probes. (B) Unique colorimetric patterns both for L. major and T. cruzi.