Detection of Entamoeba histolytica using colorimetric loop-mediated isothermal amplification (LAMP)

Sammanfattning: Amoebic dysenteri is a problem in developing countries and is caused by Entamoeba histolytica (E. histolytica) with symptoms such as diarrhea, vomiting and in worse case extra intestinal manifestation. Currently there are difficulties to diagnose E. histolytica infections in developing countries because PCR requires advanced and expensive and microscopy cannot distinguish E. histolytica from other harmless species of amoebas. The aim of this study was therefore to develop loop-mediated isothermal amplification (LAMP), which is similar to PCR but is performed at a single temperature and amplifies the target gene in less than an hour. LAMP was also compared to real time PCR. With a commercial kit, DNA were extracted from cultivated trophozoites and for the LAMP reaction, a colorimetric mastermix and six primers were used designed from 18S small subunit ribosomal RNA gene. With phenol red positive LAMP reactions showed a color change from pink to yellow and negative LAMP reactions remained pink. The sensitivity of LAMP for detection of E. histolytica was determined to be 80 pg/µl, which was ten times less sensitive than real time-PCR. The method was also shown to work on trophozoites with no DNA extraction and no non-specific amplifications were seen with DNA from G. lamblia, which showed some specificity. LAMP proved to be sensitive and easy to work with, but requires tightly closed tubes to avoid contamination and false positive results. To develop and evaluate the method LAMP for detection of E. histolytica, more studies are needed, including clinical samples and optimization.