Optimization of Colorimetric Assay of Loop Mediated Isothermal Amplification for Detections of Mycobacterium tuberculosis

Abstract

Background and objectives: Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis (MTB). It is a major public health problem of Thailand and worldwide, ranking as the second leading cause of death from worldwide infectious diseases. Diagnosis development is one of key factors to control it. The objective of this study was to optimize the colorimetric method of Loop-mediated isothermal amplification (LAMP), which is a new promising method because DNA is synthesized at a single temperature in relatively short period.  

Methodology: The LAMP was set-up to detect the genomic DNA of MTB. The amplified product was detected by using Hydroxy naphthol blue (HNB), Sybergreen (SYBR) and gel electrophoresis. The amount of HNB was optimized for detection compare to the results of using SYBR and gel electrophoreses.

Results: Pre-addition of 5 mM HNB to the LAMP reaction solution showed observe-able blue-sky color of positive but did not interfere amplification efficiency. The minimum detection limit between using HNB, SYBR and gel electrophoresis were 10, 100 and 10 pg of MTB genomic DNA, respectively.  

Conclusion: The colorimetric LAMP method using HNB was successfully optimized for detection of MTB. The optimal of concentration of HNB was 5mM and the minimum detection limit was 10 pg of MTB genomic DNA which is similar to those of gel electrophoresis.