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Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
TORCATECH LLC, 5210,104th St SW, Mukilteo, WA 98275 USA.
Florida Atlantic Univ, Charles E Schmidt Coll Med, Dept Biomed Sci, 777 Glades Rd, Boca Raton, FL 33428 USA.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, International Maternal and Child Health (IMCH), International Child Health and Nutrition. Muhimbili Univ Hlth & Allied Sci, Dept Parasitol & Med Entomol, Dar Es Salaam, Tanzania.
TORCATECH LLC, 5210,104th St SW, Mukilteo, WA 98275 USA.
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 2901Article in journal (Refereed) Published
Abstract [en]

Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) based on signal rather than target amplification under isothermal conditions was developed for nucleic acid assays. The initial signal was generated by hybridization of single stranded DNA targets to immobilized recognition probes followed by hybrid cleavage with specific restriction endonuclease (REase), and release of trigger oligonucleotides (Tr1). The signal amplification chamber contained two bead types carrying single-stranded amplification probes and two amplification REases. The probes consisted of multiple tandem repeats of either Tr1 or another trigger Tr2, with the tandem-Tr1 anchored to the beads through the antisense Tr2 linker and vice versa. Addition of the recognition reaction solution and Tr1 hybridization to the anti-Tr1 linkers started cleavage and release of additional Tr1 and Tr2, resulting in exponential signal amplification. The cleavage cascade also released horseradish peroxidase (HRP) pre-attached to the amplification probes, and the resultant signal was measured colorimetrically. A TORCA assay was developed for detection of Plasmodium falciparum parasites in blood. It had the detection limit in the attomolar concentration range, successfully detecting sub-microscopic P. falciparum infections at less than 0.75 infected erythrocytes per microliter. Further TORCA optimization will likely produce the quantitative isothermal alternative to PCR at a fraction of its cost.

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NATURE PUBLISHING GROUP , 2019. Vol. 9, article id 2901
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Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
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URN: urn:nbn:se:uu:diva-379581DOI: 10.1038/s41598-019-39921-9ISI: 000459799800050PubMedID: 30814636OAI: oai:DiVA.org:uu-379581DiVA, id: diva2:1298929
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-03-25Bibliographically approved

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Ngasala, BillyMårtensson, Andreas

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