Direct sequencing of the PCR amplified SSU rRNA gene ofEntamoeba disparand the design of primers for rapid differentiation fromEntamoeba histolytica

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Abstract
SUMMARY: Since 1993, strains ofEntamoeba histolytica sensn latohave been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains toE. histolytica sensu stricto, the non-pathogenic strains toEntamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases forE. histolytica, only a partial sequence has been published forE. dispar. Here we report a SSU rDNA sequence forE. dispar. Compared to those ofE. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from bothE. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphicDdeI restriction site which allows, after cleavage of the fragment,E. histolyticaandE. disparto be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

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