Direct amplification and differentiation of pathogenic and nonpathogenic Entamoeba histolytica DNA from stool...

Discrimination of pathogenic and nonpathogenic Entamoeba histolytica is of great clinical importance. A simple and rapid DNA extraction method that can be used directly with stool specimens was developed without the need for prior cultivation of the parasites. The entire protocol can be performed at room temperature in a 1.5-ml microcentrifuge tube format. There is no DNA precipitation step. The subsequent nested polymerase chain reaction consists of an initial E. histolytica-specific amplification, followed by two separate amplifications using two primer pairs specific for pathogenic and nonpathogenic E. histolytica, respectively. Amplification products can be verified by restriction endonuclease digests. There is no need for hybridizations or the use of radionucleotides. One trophozoite per milligram of stool sample could be detected and differentiated in a 0.1-g specimen.