Background and objectives: Tuberculosis is an
airborne disease caused by Mycobacteri um
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
Keywords
LAMP Tuberculosis HNB Loop-Mediated Isothermal Amplification Hydroxy naphthol blue