title: Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.
abstract: Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.
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: Rebeca
authors.initials: R
authors.lastname: Miranda-Castro
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: 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. mjlc@uniovi.es.
authors.forename: M Jesús
authors.initials: MJ
authors.lastname: Lobo-Castañón
mesh_terms: D001681:Biological Assay; D004265:DNA Helicases / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D055664:Electrochemical Techniques; D006801:Humans; D057230:Limit of Detection; D059346:Magnets; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D010996:Pleural Effusion / Q000382:microbiology; D015203:Reproducibility of Results; D013183:Sputum / Q000737:chemistry / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology / Q000652:urine*
chemical_list: D004269:DNA, Bacterial; D004265:DNA Helicases
keywords: Genomagnetic assay; Helicase; Isothermal amplification; Mycobacterium tuberculosis; PCR