Specific detection of Entamoeba histolytica DNA by hemolysin gene targeted PCR.
Diagnostic differentiation of pathogenic Entamoeba histolytica from non-pathogenic Entamoeba dispar is of great clinical importance. We have developed and evaluated a new polymerase chain reaction (PCR) assay (haemo-PCR) based on the novel E. histolytica hemolysin gene HLY6. The specificity of this assay was confirmed by analyzing different Entamoeba species, faeces samples, human and bacterial DNA, and digestion of amplification products with appropriate restriction enzymes. The sensitivity was confirmed by serial dilutions of E. histolytica HM-1:IMSS DNA in the excess of human DNA. Totally, 45 clinical samples were analyzed by the haemo-PCR assay including amoebic liver abscess (ALA) fluids from 23 patients suspected for amoebiasis, four faeces samples containing E. histolytica and E. dispar, and positive and negative controls. The results were compared with those obtained with PCRs for cystein-rich surface protein (P30) and small subunit ribosomal RNA (ssu rRNA) genes. The haemo-PCR gave a positive result in 18 (89%) ALA fluids compared with 14 (77%) and five (28%) by PCR for p30, and ssu rRNA, respectively. PCR products were obtained only from specimens containing E. histolytica DNA. The haemo-PCR assay was therefore found to be a valuable diagnostic tool for identification of E. histolytica infections both in faeces and ALA samples.
Department of Medicine, Unit of Infectious Diseases, Karolinska Institutet, Karolinska Hospital, 171 76, Stockholm, Sweden. sherwanzindrou@hotmail.com
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D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004748:Entamoeba histolytica / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008101:Liver Abscess, Amebic / Q000175:diagnosis / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016372:RNA, Antisense / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D016054:DNA, Protozoan; D006460:Hemolysin Proteins; D016372:RNA, Antisense; D012335:RNA, Ribosomal