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

TY - JOUR

T1 - Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

AU - Hede, Marianne Smedegaard

AU - Fjelstrup, Søren

AU - Lötsch, Felix

AU - Zoleko, Rella Manego

AU - Klicpera, Anna

AU - Groger, Mirjam

AU - Mischlinger, Johannes

AU - Endame, Lilian

AU - Veletzky, Luzia

AU - Neher, Ronja

AU - Simonsen, Anne Katrine Wrist

AU - Petersen, Eskild

AU - Mombo-Ngoma, Ghyslain

AU - Stougaard, Magnus

AU - Ho, Yi-Ping

AU - Labouriau, Rodrigo

AU - Ramharter, Michael

AU - Knudsen, Birgitta Ruth

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

AB - Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

UR - http://www.scopus.com/inward/record.url?scp=85048288590&partnerID=8YFLogxK

U2 - 10.1038/s41598-018-22378-7

DO - 10.1038/s41598-018-22378-7

M3 - Journal article

C2 - 29515150

SN - 2045-2322

VL - 8

SP - 4122

JO - Scientific Reports

JF - Scientific Reports

IS - 1

M1 - 4122

ER -