Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device

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 10(3) TB cells/mL of sputum at 53.5 +/- 3.3 minutes, 10(4) cells/mL at 46.3 +/- 2.2 minutes, and 10(5) 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 10(3) cells/mL at 74 +/- 10 minutes, 10(4) cells/mL at 60 +/- 9 minutes, and 10(5) 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 Opti-Gene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 +/- 3.3 minutes, 10(4) cells/mL at 45.4 +/- 11.3 minutes, and 10(5) 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.