Development of loop-mediated isothermal amplification assay for detection of Entamoeba histolytica

Abstract

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.

FIG. 1.

Sensitivity of the E. histolytica LAMP versus that of nested PCR. The E. histolytica LAMP and nested PCR were carried out at concentrations ranging from 10³ to 10⁻³ parasites per reaction. (A) Visual detection under UV light of LAMP products in the serial 10-fold dilutions of E. histolytica DNA. (B) Sensitivity of electrophoretic analysis of the E. histolytica LAMP products. (C) Sensitivity of electrophoretic analysis of nested PCR-amplified products.

FIG. 2.

The specificity of the E. histolytica LAMP was determined with DNA extracted from four different Entamoeba spp. and Giardia lamblia. Lanes 1 and 7, E. histolytica strains Rahman and HM-1:IMSS; lane 2, E. moshkovskii; lane 3, E. dispar; lane 4, E. invadens; lane 5, Giardia lamblia; lane 6, negative control (water alone). The LAMP products were visualized with calcein fluorescent detection reagent under UV light (lower panel) or electrophoresed in 2% gel and stained with ethidium bromide (upper panel).