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Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica

Published online by Cambridge University Press:  06 April 2009

S. Novati ,
M. Sironi ,
S. Granata ,
A. Bruno ,
S. Gatti ,
M. Scaglia  and
C. Bandi
S. Novati
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
M. Sironi
Affiliation:
Istituto di Patologia Generate Veterinaria, Università di Milano, Via Celoria 10, 20133 Milano, Italy
S. Granata
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy
A. Bruno
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
S. Gatti
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
M. Scaglia
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
C. Bandi*
Affiliation:
Istituto di Patologia Generate Veterinaria, Università di Milano, Via Celoria 10, 20133 Milano, Italy Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy
*
* Corresponding author: Dr C. Bandi, Istituo di Patologia Generale Veterinaria, Università Milano, Via Celoria 10, 20133 Milano, Italy.
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Summary

Since 1993, strains of Entamoeba histolytica sensn 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.

Keywords

Entamoeba disparEntamoeba histolyticaribosomal RNAspecies identificationRFLPdirect sequencing
Type
Research Article
Information
Parasitology , Volume 112 , Issue 4 , April 1996 , pp. 363 - 369
Copyright
Copyright © Cambridge University Press 1996

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