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

Abstract

Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba 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 for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. 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 both E. 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 polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Amebic infections due to the Entamoeba histolytica-Entamoeba dispar complex: a study of the incidence in a remote rural area of Ecuador.

“…Unfortunately, they are difficult to perform and timeconsuming. Several biomolecular techniques are available, based on the amplification of different rRNA genes of the two relative species, 18,24,25 but most of them cannot be performed in field studies because they require expensive technical support, chemicals, and well-trained personnel.…”

Comparison of Real-Time PCR Protocols for Differential Laboratory Diagnosis of Amebiasis

“…Several PCR assays designed for differential detection of E. histolytica and E. dispar have been developed. Most of them target either the small-subunit rRNA (18S rRNA) gene (5,10,15,18) or species-specific episomal repeats (1,19,21). These targets are present on multicopy, extrachromosomal plasmids in the amebas (3).…”

Entamoeba histolytica

“…Of all the different gene targets used to differentiate between E. histolytica and E. dispar by PCR based assays (Sequence of the extra-chromosomal circular rRNA gene, the repetitive sequences in the non coding regions of rDNA, 16S rRNA genes), the small subunit rRNA gene is found to be more sensitive than the best antigen detection method in stool culture (Novati et al, 1996;Mirelman et al, 1997). It is in wide use as it is present in multiple copies on the extra-chromosomal plasmids (Bhattacharya et al, 1988;.…”