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Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi
Fatemeh Mahdavi1, Hamed Mirjalali2, Maryam Niyyati1
1Department of Medical Parasitology and Mycology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Abstract:
Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi -positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/μL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.
Insights
A new loop-mediated isothermal amplification (LAMP) assay was developed for detecting Enterocytozoon bieneusi, a common cause of microsporidiosis. While highly specific, the assay detected 83.3% of positive samples, making it a promising screening tool.
Area of Science:
- Microbiology
- Parasitology
- Molecular Diagnostics
Background:
- Enterocytozoon bieneusi is a leading cause of microsporidiosis in humans and animals, often linked to waterborne and foodborne outbreaks.
- Rapid and accurate detection of E. bieneusi is critical for clinical diagnosis and public health surveillance.
- Existing diagnostic methods may require specialized equipment or lengthy procedures.
Purpose of the Study:
- To develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of E. bieneusi.
- To assess the sensitivity, specificity, and efficiency of the developed LAMP assay.
- To compare the performance of the LAMP assay with real-time PCR.
Main Methods:
- LAMP primers were designed targeting the small subunit ribosomal RNA (SSU rRNA) gene of E. bieneusi.
- DNA was extracted from 30 E. bieneusi-positive samples previously confirmed by nested PCR.
- LAMP reactions were conducted at 63°C for 60 minutes, and results were compared with real-time PCR.
Main Results:
- The LAMP assay successfully amplified 83.3% (25/30) of E. bieneusi-positive samples.
- The assay demonstrated high specificity, with no cross-reactivity observed with other microorganisms.
- The limit of detection (LoD) for the LAMP assay was as low as 34 ag/μL of total DNA.
Conclusions:
- The developed LAMP assay offers a rapid, specific, and instrument-independent method for E. bieneusi detection.
- While not as sensitive as real-time PCR in this study, its advantages make it suitable for initial screening and outbreak investigations.
- Further optimization may enhance the sensitivity of the LAMP assay for broader clinical application.
