Related Experiment Video
Updated: May 5, 2026
The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii
Published on: April 18, 2016
Hydroxynaphthol blue-based loop-mediated isothermal amplification for the rapid detection of Burkholderia
Wiphat Klayut1, Panatda Aramrueang2, Benjawan Phetsuksiri1,3
1National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi Province, 11000, Thailand.
Background:
Melioidosis is a potentially life-threatening disease caused by Burkholderia pseudomallei. It is endemic in Southeast Asia and northern Australia and is increasingly distributed in other tropical and subtropical regions. Fast and accurate detection of B. pseudomallei is crucial for initiating prompt and effective treatment.
Methods:
Loop-mediated isothermal amplification (LAMP) using hydroxynaphthol blue (HNB) for visual detection has been developed and evaluated to detect B. pseudomallei.
Results:
The assay amplified B. pseudomallei TTSS1-orf2 at 63°C with a 60-min reaction and no cross-amplification was observed. The detection limit was 1.85×102 fg/µl of B. pseudomallei DNA or 2.08×102 CFU/ml of B. pseudomallei spiked in blood, sputum and urine samples. Clinical evaluation using 191 residual specimens revealed 100% negative agreement in detecting 48 of 49 culture-positive samples. Overall, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value were 97.96%, 100%, 99.48%, 100% and 99.30%, respectively, while the κ index showed substantial agreement with the culture method (κ=0.99).
Conclusions:
This LAMP assay demonstrated good performance, with easy operation and low cost for the rapid detection of B. pseudomallei. It has the potential to be an alternative molecular tool for the early diagnosis of melioidosis in clinical settings.
