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Home / Equine Research Bank / BMC Microbiol / A novel ready-to-use loop-mediated isothermal amplification ...
BMC microbiology2025; 25(1); 36; doi: 10.1186/s12866-024-03737-z

A novel ready-to-use loop-mediated isothermal amplification (LAMP) method for detection of Burkholderia mallei and B. pseudomallei.

Abstract: 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.
© 2025. The Author(s).
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Publication Date: 2025-01-21 PubMed ID: 39838326PubMed Central: PMC11749068DOI: 10.1186/s12866-024-03737-zGoogle Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research developed a new, simple, and ready-to-use dried LAMP method to detect the bacteria Burkholderia mallei and Burkholderia pseudomallei, which cause the zoonotic diseases glanders and melioidosis.
  • The new diagnostic tool is designed to be cost-effective, rapid, and sensitive, making it easier to diagnose infections in resource-limited areas compared to traditional PCR and bacterial isolation methods.

Background

  • Diseases: Glanders and melioidosis are contagious diseases transmitted from animals to humans (zoonotic), caused by Burkholderia mallei and Burkholderia pseudomallei respectively.
  • Current Diagnostic Challenges:
    • Bacterial isolation and PCR are standard diagnostic methods.
    • Both require skilled technicians and expensive laboratory equipment.
    • Such requirements limit access to effective diagnosis in low-resource or remote areas.

Method Development

  • Loop-Mediated Isothermal Amplification (LAMP): A nucleic acid amplification technique performed at a constant temperature, which is simpler and faster than PCR.
  • Primer Design: Primers were developed targeting the internal transcribed spacer (ITS) region specific to B. mallei and B. pseudomallei DNA, ensuring selective detection.
  • Dried-Format LAMP Assay: The team created a ready-to-use dried reagent format to facilitate practical field use without requiring complex preparation or refrigeration.

Experimental Results

  • Sensitivity: The assay detected as few as 10 genomic copies of B. mallei and B. pseudomallei DNA using liquid LAMP.
  • Specificity: The primers did not amplify DNA from other Burkholderia species, demonstrating high specificity for the target bacteria.
  • Testing on Clinical Samples:
    • Applied to nasal and skin swabs from 19 infected horses and 5 uninfected control horses.
    • Compared results with real-time PCR for validation.
    • The dried LAMP assay agreed with PCR results for 87.5% of the positives and 100% of the negatives.
    • All tested B. pseudomallei clinical isolates were detected by the novel assay.

Conclusions and Implications

  • This study reports the first dried LAMP assay for detecting B. mallei and B. pseudomallei, representing a significant advancement over previous diagnostic methods.
  • The dried LAMP format is simple, rapid, cost-effective, and sensitive, which enhances feasibility for on-site or field diagnostics.
  • This tool can greatly improve disease monitoring and control of glanders and melioidosis in areas with limited laboratory infrastructure.
  • Its use could facilitate quicker diagnosis and treatment decisions, ultimately aiding in better disease management and reducing zoonotic transmission.

Cite This Article

APA
Nakase M, Thapa J, Batbaatar V, Khurtsbaatar O, Enkhtuul B, Unenbat J, Lkham B, Fujita S, Koshikawa A, Tuanyok A, Saechan V, Higashi H, Hayashida K, Suzuki Y, Nakajima C, Kimura T. (2025). A novel ready-to-use loop-mediated isothermal amplification (LAMP) method for detection of Burkholderia mallei and B. pseudomallei. BMC Microbiol, 25(1), 36. https://doi.org/10.1186/s12866-024-03737-z

Publication

BMC microbiology
ISSN: 1471-2180
NlmUniqueID: 100966981
Country: England
Language: English
Volume: 25
Issue: 1
Pages: 36
PII: 36

Researcher Affiliations

Nakase, Mitsuru
  • Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-Ku, Sapporo, Hokkaido, 060-0818, Japan.
Thapa, Jeewan
  • Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Kita 20 Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan.
Batbaatar, Vanaabaatar
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Zaisan 17024, Khan-Uul District, Ulaanbaatar City, Ulaanbaatar, Mongolia.
Khurtsbaatar, Ochirbat
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Zaisan 17024, Khan-Uul District, Ulaanbaatar City, Ulaanbaatar, Mongolia.
Enkhtuul, Batchuluun
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Zaisan 17024, Khan-Uul District, Ulaanbaatar City, Ulaanbaatar, Mongolia.
Unenbat, Jugderkhorloo
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Zaisan 17024, Khan-Uul District, Ulaanbaatar City, Ulaanbaatar, Mongolia.
Lkham, Baasansuren
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Zaisan 17024, Khan-Uul District, Ulaanbaatar City, Ulaanbaatar, Mongolia.
Fujita, Sachiho
  • Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-Ku, Sapporo, Hokkaido, 060-0818, Japan.
Koshikawa, Ai
  • Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-Ku, Sapporo, Hokkaido, 060-0818, Japan.
Tuanyok, Apichai
  • Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16Th Ave Gainesville, Florida, 32608, USA.
Saechan, Vannarat
  • Faculty of Veterinary Science, Prince of Songkla University, Poonnakan Rd, Kho Hong, Hat Yai District, Songkhla, 90110, Thailand.
Higashi, Hideaki
  • Division of Infection and Immunity, International Institute for Zoonosis Control, Hokkaido University, Kita 20 Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan.
Hayashida, Kyoko
  • Division of Collaborations and Education, International Institute for Zoonosis Control, Hokkaido University, Kita 20 Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan.
Suzuki, Yasuhiko
  • Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Kita 20 Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan.
  • Institute for Vaccine Research and Development, Hokkaido University, Kita 21 Nishi 11, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan.
Nakajima, Chie
  • Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Kita 20 Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan.
  • Institute for Vaccine Research and Development, Hokkaido University, Kita 21 Nishi 11, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan.
Kimura, Takashi
  • Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-Ku, Sapporo, Hokkaido, 060-0818, Japan. tkimura@vetmed.hokudai.ac.jp.

MeSH Terms

  • Burkholderia mallei / isolation & purification
  • Burkholderia mallei / genetics
  • Burkholderia pseudomallei / isolation & purification
  • Burkholderia pseudomallei / genetics
  • Nucleic Acid Amplification Techniques / methods
  • Animals
  • Melioidosis / diagnosis
  • Melioidosis / veterinary
  • Melioidosis / microbiology
  • Glanders / diagnosis
  • Glanders / microbiology
  • Sensitivity and Specificity
  • Molecular Diagnostic Techniques / methods
  • DNA, Bacterial / genetics
  • Horses
  • DNA Primers / genetics
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology

Grant Funding

  • JP23jm0110021 / Japan Agency for Medical Research and Development

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: This study was approved by the Animal Ethics Committee of the Veterinary Science and Bio-medical Research at the Mongolian University of Life Science (VSBMR23/01/21) and was carried out in accordance with the Standard Diagnosis Guide for Animal Diseases 2015 of the State Central Veterinary Laboratory, Mongolia. In accordance with the General Authority for Veterinary Services of Mongolia, no approval was required for ante-mortem sampling as it was conducted as part of standard veterinary care. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

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Citations

This article has been cited 1 times.
  1. Gassiep I, Chewapreecha C, Chantratita N, Oakley T, Mukhopadhyay C, Lal PB, AuCoin D, Chowdhury FR, Meumann EM, Currie BJ, Dance DAB, Wuthiekanun V, Norton R. Laboratory diagnosis of melioidosis.. PLoS Negl Trop Dis 2025 Dec;19(12):e0013761.
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