title: Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.
abstract: A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.
authors.affiliation: Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Barreda-García
authors.affiliation: Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: González-Álvarez
authors.affiliation: Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: de-Los-Santos-Álvarez
authors.affiliation: Servicio de Microbiología, Unidad de Referencia Regional de Micobacterias de Asturias. Hospital Universitario Central de Asturias, Avda Roma s/n, 33011 Oviedo, Spain.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Palacios-Gutiérrez
authors.affiliation: Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: Arturo J
authors.initials: AJ
authors.lastname: Miranda-Ordieres
authors.affiliation: Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: mjlc@uniovi.es.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Lobo-Castañón
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D004265:DNA Helicases / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D013183:Sputum / Q000737:chemistry / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004265:DNA Helicases
keywords: Electrochemical biosensor; Helicase; Hybridization assay; Isothermal amplification; Mycobacterium tuberculosis