Journal: Scientific Reports

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

doi: 10.1038/s41598-019-39946-0

Figure Lengend Snippet: ( A ) Multiple sequences alignment of L . major vs T . cruzi . Forward (highlighted in yellow) and reverse (highlighted in green) primers. Respectively, highlighted in red and blue, the abasic PNA 1 and PNA 2 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 PNA 1 probe (emphasized also with a red rectangle), while the abasic PNA 2 probe interrogates the first SNF (SNF 1 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 PNA 1 and PNA 2 , represented with the red or blue question mark respectively. ( C ) Each parasite gives rise to a specific SMART-C-Biotin incorporation pattern. T . cruzi gives a positive result for both abasic probes, whereas L . major only shows a positive result in one, abasic PNA 1 .

Article Snippet: The two abasic PNA probes (Abasic PNA 1 and PNA 2 ) were synthesized by DestiNA Genomica SL (Spain) using standard solid-phase synthesis techniques on an Intavis Bioanalytical Instruments MultiPrep CF Synthesizer (Intavis AG GmH, Germany).

Techniques: Amplification, Sequencing

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-39946-0

Figure Lengend Snippet: ( 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 PNA 2 ; in red, 3 spots of abasic PNA 1 .

Article Snippet: The two abasic PNA probes (Abasic PNA 1 and PNA 2 ) were synthesized by DestiNA Genomica SL (Spain) using standard solid-phase synthesis techniques on an Intavis Bioanalytical Instruments MultiPrep CF Synthesizer (Intavis AG GmH, Germany).

Techniques:

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-39946-0

Figure Lengend Snippet: ( A ) Spotting layout with 3 Biotin marker spots (yellow) rather than the 2 of Fig. . 3 spots of PNA 1 (red) and 3 spots of PNA 2 (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.

Article Snippet: The two abasic PNA probes (Abasic PNA 1 and PNA 2 ) were synthesized by DestiNA Genomica SL (Spain) using standard solid-phase synthesis techniques on an Intavis Bioanalytical Instruments MultiPrep CF Synthesizer (Intavis AG GmH, Germany).

Techniques: Marker, Electrophoresis, Produced, Software, Negative Control, Concentration Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-39946-0

Figure Lengend Snippet: ( 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 SNF 1 for templating the SMART-C-Biotin incorporation respectively in the abasic PNA 1 and PNA 2 probes. ( B ) Unique colorimetric patterns both for L . major and T . cruzi .

Article Snippet: The two abasic PNA probes (Abasic PNA 1 and PNA 2 ) were synthesized by DestiNA Genomica SL (Spain) using standard solid-phase synthesis techniques on an Intavis Bioanalytical Instruments MultiPrep CF Synthesizer (Intavis AG GmH, Germany).

Techniques: