title: Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.
abstract: Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.
authors.affiliation: Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil.
authors.forename: Thiago Dos Santos
authors.initials: Tdos S
authors.lastname: Gomes
authors.affiliation: Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.
authors.forename: Mariana Coimbra
authors.initials: MC
authors.lastname: Garcia
authors.affiliation: Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil.
authors.forename: Flavia
authors.initials: F
authors.lastname: de Souza Cunha
authors.affiliation: Faculdade de Medicina, Universidade Federal Fluminense, 24030-210 Niterói, RJ, Brazil.
authors.forename: Heloisa
authors.identifier: 0000-0002-4440-0546
authors.initials: H
authors.lastname: Werneck de Macedo
authors.affiliation: Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Peralta
authors.affiliation: Faculdade de Medicina, Universidade Federal Fluminense, 24030-210 Niterói, RJ, Brazil ; Departamento de Patologia, Hospital Universitário Antônio Pedro, Rua Marquês do Paraná 303, Prédio Principal, 4° andar, Centro, 24030-210 Niterói, RJ, Brazil.
authors.forename: Regina Helena Saramago
authors.initials: RH
authors.lastname: Peralta
mesh_terms: D003937:Diagnosis, Differential; D004747:Entamoeba / Q000145:classification / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D009930:Organic Chemicals; D057089:Pathology, Molecular; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D009930:Organic Chemicals; C474470:SYBR Gold nucleic acid gel stain