Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube

Abstract

Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65°C and 60min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.