Optimization of Loop-Mediated Isothermal Amplification (LAMP) Conditions for the Detection of Mycobacterium tuberculosis complex
DOI:
10.29303/jbt.v26i4.13826Published:
2026-11-02Downloads
Abstract
Tuberculosis (TB) remains a major global health concern, highlighting the need for accessible molecular diagnostic methods. Loop-mediated isothermal amplification (LAMP) is a DNA amplification technique with potential for TB detection, although its performance depends on appropriate reaction conditions. Objective: This study aimed to evaluate incubation temperature and time for LAMP amplification targeting the mpt64 (Rv1980c) gene. This laboratory experimental study was conducted at the Center for Diagnostic Research and Infectious Diseases Laboratory, Universitas Andalas, Padang, Indonesia. Nine temperature–time combinations were evaluated using three incubation temperatures (63°C, 64°C, and 65°C) and three incubation times (30, 40, and 60 minutes). Amplification results were assessed using SYBR Green I fluorescence and 2% agarose gel electrophoresis and analyzed descriptively. The condition of 65°C for 60 minutes was selected for further evaluation because a clearer DNA band was observed in the positive control, whereas no clear DNA band was detected in the negative control. However, green fluorescence was observed in both controls in some observations, indicating inconsistent visual differentiation using SYBR Green I. The reaction condition of 65°C for 60 minutes was selected as the most suitable condition among those evaluated based on the agarose gel electrophoresis results. Further studies are needed to assess the consistency and specificity of the amplification products.
Keywords:
LAMP mpt64 Reaction optimization SYBR Green I TuberculosisReferences
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