title: Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.
abstract: In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting.
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Creecy
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America.
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Russ
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America.
authors.forename: Francesca
authors.initials: F
authors.lastname: Solinas
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, United States of America.
authors.forename: David W
authors.initials: DW
authors.lastname: Wright
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America; Department of Chemistry, Vanderbilt University, Nashville, TN, United States of America.
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Haselton
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013183:Sputum / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial