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Updated: Nov 3, 2025
Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens
Published on: February 20, 2015
Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP)
T Sathish Kumar1, K Radhika1, J Joseph Sahaya Rajan1
1ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India.
Abstract:
Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.
Insights
A new visual Loop-mediated isothermal amplification (LAMP) assay offers rapid and accurate detection of Enterocytozoon hepatopenaei (EHP), a significant shrimp pathogen. This user-friendly method is ideal for on-site diagnosis in aquaculture settings.
Area of Science:
- Aquaculture pathology
- Molecular diagnostics
- Microbiology
Background:
- Hepatopancreatic microsporidiosis (HPM), caused by Enterocytozoon hepatopenaei (EHP), is a major threat to shrimp aquaculture globally.
- Effective disease management relies on early, rapid, and accurate diagnostic methods to mitigate economic losses.
Purpose of the Study:
- To develop an improved, simple, closed-tube, colorimetric Loop-mediated isothermal amplification (LAMP) assay for the visual detection of EHP.
- To validate the diagnostic performance of the developed LAMP assay in various clinical samples.
Main Methods:
- A novel LAMP assay was designed using primers specific to the EHP spore wall protein (SWP) gene.
- Visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the reaction tubes.
- The assay was tested on diverse clinical samples including hepatopancreas, FTA cards, feces, pond water, and soil.
Main Results:
- The visual LAMP assay demonstrated 100% diagnostic sensitivity and 100% diagnostic specificity in clinical evaluations.
- The assay successfully detected EHP in all tested sample types.
- EHP DNA elution from FTA cards was achieved in just 17 minutes using a dry bath.
Conclusions:
- The developed visual LAMP assay provides a rapid (within 40 minutes), user-friendly, and cost-effective method for EHP detection.
- This assay requires no specialized equipment or technical expertise, making it suitable for farm-site application in resource-limited settings.
