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.

An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies

“…Interestingly, extraction and LAMP detection have been integrated in single devices for detection of e.g. Mycobacterium tuberculosis in sputum samples [54]. Whether isolation of O. volvulus DNA from skin biopsies also can be achieved with high efficiency using similar extraction methods, will need to be further investigated.…”

Point-of-Care Diagnostics: Recent Developments in a Connected Age

“…A self-contained sample preparation technique integrated with LAMP has been demonstrated 233 . A nested PCR device for detection of bacterial pathogen, with integrated sample preparation from serum samples, has been shown 234 , although the serum must first be isolated.…”

A Prototype Biomarker Detector Combining Biomarker Extraction and Fixed Temperature PCR

“…6,12 Target biomarkers consisted of 140 base pair fragments of the IS6110 sequence of tuberculosis. 6 A 67 bp amplicon of the IS6110 sequence was amplified using 0.2 µM forward primer (5′ ACC AGC ACC TAA CCG GCT GTG G 3′) and 0.2 µM reverse primer (5′ CAT CGT GGA AGC GAC CCG CCA G 3′). 12 Chamber 1 ( Fig.…”