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Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei
1Microbiology Division, Defence Research and Development Establishment, Gwalior, India.
Abstract:
Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103 CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.
Insights
A new real-time loop-mediated isothermal amplification (LAMP) assay offers rapid and specific detection of Burkholderia mallei, the cause of glanders. This sensitive molecular diagnostic tool aids early diagnosis and control of this re-emerging zoonotic disease.
Area of Science:
- Microbiology
- Molecular Biology
- Veterinary Medicine
Background:
- Glanders, caused by Burkholderia mallei, is a contagious zoonotic disease requiring early diagnosis for effective treatment and control.
- Accurate molecular detection of B. mallei is challenging due to its genetic similarity to Burkholderia pseudomallei.
Purpose of the Study:
- To develop a simple, rapid, specific, and sensitive molecular assay for the detection of Burkholderia mallei.
- To provide a viable alternative to PCR-based methods, especially in resource-limited settings.
Main Methods:
- Design of six B. mallei-specific primers.
- Development and validation of a real-time loop-mediated isothermal amplification (LAMP) assay.
- Testing sensitivity using B. mallei genomic DNA and spiked human blood samples.
Main Results:
- The developed LAMP assay demonstrated high sensitivity, detecting as low as 1 pg of B. mallei genomic DNA.
- The assay successfully detected B. mallei at concentrations of 5.5 × 10^3 CFU/ml in spiked human blood.
- The assay exhibited high specificity for B. mallei, with no cross-reactivity observed with other bacterial strains.
Conclusions:
- The real-time LAMP assay is a sensitive, specific, and rapid method for detecting Burkholderia mallei.
- This field-adaptable assay presents a practical alternative to PCR for B. mallei detection in glanders-endemic regions, particularly those with limited resources.
