title: Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.
abstract: This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.
authors.affiliation: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Carinelli
authors.affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Box 815, 751 08 Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Kühnemund
authors.affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Box 815, 751 08 Uppsala, Sweden; Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Box 1031, Se-171 21 Solna, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Spain. Electronic address: isabel.pividori@uab.cat.
authors.forename: M I
authors.initials: MI
authors.lastname: Pividori
mesh_terms: D015374:Biosensing Techniques; D018076:DNA, Complementary / Q000235:genetics; D029043:Ebolavirus / Q000235:genetics / Q000302:isolation & purification*; D019142:Hemorrhagic Fever, Ebola / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D006801:Humans; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D012324:RNA-Dependent RNA Polymerase
keywords: Circle-to-circle amplification; Ebola virus; Electrochemical genosensing; Isothermal amplification; Magnetic particle