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A novel ready-to-use loop-mediated isothermal amplification (LAMP) method for detection of Burkholderia mallei and B.
Mitsuru Nakase1, Jeewan Thapa2, Vanaabaatar Batbaatar3
1Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-Ku, Sapporo, Hokkaido, 060-0818, Japan.
Background:
Glanders and melioidosis are contagious zoonotic diseases caused by Burkholderia mallei and B. pseudomallei, respectively. Bacterial isolation and polymerase chain reaction (PCR) have been used to detect these bacteria in animals suspected of infection; however, both methods require skilled experimental techniques and expensive equipment. These obstacles make it difficult to diagnose B. mallei and B. pseudomallei infections in areas where reagents and equipment are difficult to procure. To solve this problem, we developed an easy and ready-to-use dried-format diagnostic tool based on loop-mediated isothermal amplification (LAMP) method.
Results:
The primer set targeting the internal transcribed spacer (ITS) region detected 10 genomic copies of B. mallei DNA and B. pseudomallei DNA using the conventional liquid LAMP method. This primer set did not detect any other Burkholderia species. Using this novel primer set, a dried-format in-house LAMP method with high sensitivity and specificity was developed. This method was used to test for the presence of B. mallei DNA in swabs collected from the nasal cavity and ulcerated skin of 19 B. mallei-infected horses and five uninfected horses and was compared with the real-time PCR method. These two tests showed 87.5% agreement for the positive samples and 100% agreement for the negative samples. This method detected all tested B. pseudomallei clinical isolates.
Conclusions:
We established the first dry LAMP method for the detection of B. mallei and B. pseudomallei. This study provided a simple, rapid, cost-effective, and sensitive diagnostic tool for glanders and melioidosis.
Insights
A new dried-format loop-mediated isothermal amplification (LAMP) assay offers a simple, rapid, and cost-effective method for detecting Burkholderia mallei and B. pseudomallei, aiding in the diagnosis of glanders and melioidosis.
Area of Science:
- Veterinary Microbiology
- Diagnostic Microbiology
- Molecular Diagnostics
Background:
- Glanders and melioidosis are zoonotic diseases caused by Burkholderia mallei and B. pseudomallei.
- Current diagnostic methods like bacterial isolation and PCR are resource-intensive, hindering diagnosis in resource-limited settings.
- There is a need for accessible diagnostic tools for these diseases.
Purpose of the Study:
- To develop a novel, easy-to-use, dried-format diagnostic tool for detecting B. mallei and B. pseudomallei.
- To establish a sensitive and specific assay for glanders and melioidosis detection.
Main Methods:
- Development of a dried-format loop-mediated isothermal amplification (LAMP) assay.
- Design of a primer set targeting the internal transcribed spacer (ITS) region of B. mallei and B. pseudomallei.
- Validation of the assay using clinical samples from infected and uninfected horses and B. pseudomallei isolates.
Main Results:
- The primer set specifically detected 10 genomic copies of B. mallei and B. pseudomallei DNA, with no cross-reactivity with other Burkholderia species.
- The dried-format LAMP assay demonstrated high sensitivity and specificity, with 87.5% agreement with real-time PCR for B. mallei detection in horses.
- The assay successfully detected all tested B. pseudomallei clinical isolates.
Conclusions:
- The study established the first dry LAMP method for detecting B. mallei and B. pseudomallei.
- This novel diagnostic tool is simple, rapid, cost-effective, and sensitive.
- The developed assay provides a valuable resource for diagnosing glanders and melioidosis, particularly in resource-limited areas.
