Journal: Scientific Reports

Article Title: Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

doi: 10.1038/s41598-018-22378-7

Figure Lengend Snippet: Detection of pTopI using a colorimetric readout. ( A ) The individual steps of the colorimetric readout are schematically depicted. (I) shows the RCA primer hybridized to the pTopI generated single stranded DNA circles. II) The primer is elongated in a RCA reaction performed in the presence of biotin labeled nucleotides (red asterisks). III) The biotinylated DNA generated is bound to a silica membrane, which is positioned in a column. IV) Silica bound biotinylated DNA is visualized by coupling to streptavidin conjugated HRP (black asterisks) followed by V) incubation with the colorimetric HRP substrate, TMB. ( B ) Representative pictures of the silica membranes after completion of the colorimetric readout using either a sample without DNA circles or spiked with premade DNA circles (F1 and F2 respectively) as well as after testing saliva samples from uninfected individuals (F3 and F4) or Plasmodium infected individuals (F5 and F6) (patients #4 and #20 respectively, see Table ). ( C ) Box plot representing the result of using the above described colorimetric readout for testing 14 saliva samples from uninfected individuals and 16 samples from Plasmodium infected individuals. The results are shown as fold increase above the average signal obtained from the analysis of samples from uninfected individuals.

Article Snippet: Finally, 150 µL TMB Enhanced HRP Membrane Substrate (ESPM) (Surmodics) was added to the columns.

Techniques: Generated, Labeling, Membrane, Incubation, Infection