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Updated: Jul 3, 2026
Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens
Published on: February 20, 2015
A rapid and specific loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting
Anocha Poommouang1, Phanupong Changtor1, Pakjira Tongsut2
1Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand.
Abstract:
Melissococcus plutonius, a gram-positive bacterium, causes European Foulbrood (EFB) disease in honeybee larvae, leading to reduced yields in various countries. This study developed a rapid detection method for M. plutonius using colorimetric loop-mediated isothermal amplification with a lateral flow dipstick (cLAMP-LFD), based on the sequences of gyrase subunit B (gyrB) and superoxide dismutase (sodA) genes, giving three primer sets (gyrB1, gyrB2, and sodA). The cLAMP-LFD using gyrB1 was capable of detecting M. plutonius specifically and sensitively under the optimal condition of 63°C for 50 min with no cross-reactivity with other bacteria, and the limit of detection (LOD) was 2 × 104 copies per reaction and 50 CFU of bacterial cells per reaction. In spiked samples, the assay successfully detected M. plutonius in honeybee larvae and adult honeybees, with minimum detectable levels of 11.4 and 80 CFU per reaction, respectively. Our results demonstrate that the gyrB based 1cLAMP-LFD assay is suitable for detecting M. plutonius in apiary settings and in commercial honeybee samples, including those that tested negative by conventional methods. Overall, this cLAMP-LFD assay represents a rapid, sensitive, and specific tool for M. plutonius detection and may facilitate field surveillance, disease control, and management of EFB outbreaks worldwide, thereby supporting improved honey production efficiency.
Insights
A new colorimetric loop-mediated isothermal amplification with a lateral flow dipstick (cLAMP-LFD) assay rapidly detects Melissococcus plutonius, the cause of European Foulbrood (EFB). This sensitive method aids in early disease detection and management for honeybee health.
Area of Science:
- Microbiology and Molecular Diagnostics
- Apiculture and Veterinary Entomology
Background:
- Melissococcus plutonius causes European Foulbrood (EFB), a significant honeybee larval disease impacting global honey production.
- Current detection methods for M. plutonius can be time-consuming, limiting rapid field diagnosis and intervention.
Purpose of the Study:
- To develop and validate a rapid, sensitive, and specific diagnostic tool for M. plutonius detection.
- To enable efficient surveillance and management of European Foulbrood disease in apiaries.
Main Methods:
- Development of a colorimetric loop-mediated isothermal amplification with a lateral flow dipstick (cLAMP-LFD) assay targeting gyrase subunit B (gyrB) and superoxide dismutase (sodA) genes.
- Optimization of the gyrB1 primer set for specific and sensitive detection under isothermal conditions (63°C for 50 min).
- Validation using bacterial cell cultures and spiked honeybee samples (larvae and adults).
Main Results:
- The gyrB1-based cLAMP-LFD assay demonstrated high specificity, with no cross-reactivity against other common bacteria.
- The limit of detection was as low as 2 × 10⁴ copies/reaction and 50 CFU/reaction for pure cultures.
- Successful detection of M. plutonius in spiked honeybee larvae (11.4 CFU/reaction) and adult bees (80 CFU/reaction), outperforming conventional methods in some cases.
Conclusions:
- The gyrB-based cLAMP-LFD assay is a rapid, sensitive, and specific tool for detecting M. plutonius in apiary and commercial settings.
- This assay facilitates timely field surveillance, disease control, and management of EFB outbreaks, supporting honey production efficiency.
- The developed method offers a valuable alternative for diagnosing EFB, especially in samples that test negative via traditional techniques.
