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The Scientific World Journal
Volume 2014, Article ID 645084, 8 pages
http://dx.doi.org/10.1155/2014/645084
Research Article

Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

1Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil
2Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
3Faculdade de Medicina, Universidade Federal Fluminense, 24030-210 Niterói, RJ, Brazil
4Departamento de Patologia, Hospital Universitário Antônio Pedro, Rua Marquês do Paraná 303, Prédio Principal, 4° andar, Centro, 24030-210 Niterói, RJ, Brazil

Received 5 November 2013; Accepted 29 December 2013; Published 12 February 2014

Academic Editors: J. P. Ackers and H. Hooshyar

Copyright © 2014 Thiago dos Santos Gomes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 ( ) was 73°C and 70°C, respectively. For E. hartmanni, the 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.