Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction
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 m...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2014-01-01
|
| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1155/2014/645084 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832558915698556928 |
|---|---|
| author | Thiago dos Santos Gomes Mariana Coimbra Garcia Flavia de Souza Cunha Heloisa Werneck de Macedo José Mauro Peralta Regina Helena Saramago Peralta |
| author_facet | Thiago dos Santos Gomes Mariana Coimbra Garcia Flavia de Souza Cunha Heloisa Werneck de Macedo José Mauro Peralta Regina Helena Saramago Peralta |
| author_sort | Thiago dos Santos Gomes |
| collection | DOAJ |
| description | 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 (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm 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. |
| format | Article |
| id | doaj-art-230ab43d9a984d67b1dad2c46fa8d80b |
| institution | Kabale University |
| issn | 2356-6140 1537-744X |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | The Scientific World Journal |
| spelling | doaj-art-230ab43d9a984d67b1dad2c46fa8d80b2025-02-03T01:31:14ZengWileyThe Scientific World Journal2356-61401537-744X2014-01-01201410.1155/2014/645084645084Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain ReactionThiago dos Santos Gomes0Mariana Coimbra Garcia1Flavia de Souza Cunha2Heloisa Werneck de Macedo3José Mauro Peralta4Regina Helena Saramago Peralta5Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, BrazilInstituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, BrazilDepartamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, BrazilFaculdade de Medicina, Universidade Federal Fluminense, 24030-210 Niterói, RJ, BrazilInstituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, BrazilFaculdade de Medicina, Universidade Federal Fluminense, 24030-210 Niterói, RJ, BrazilAmoebiasis, 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 (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm 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.http://dx.doi.org/10.1155/2014/645084 |
| spellingShingle | Thiago dos Santos Gomes Mariana Coimbra Garcia Flavia de Souza Cunha Heloisa Werneck de Macedo José Mauro Peralta Regina Helena Saramago Peralta Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction The Scientific World Journal |
| title | Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction |
| title_full | Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction |
| title_fullStr | Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction |
| title_full_unstemmed | Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction |
| title_short | Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction |
| title_sort | differential diagnosis of entamoeba spp in clinical stool samples using sybr green real time polymerase chain reaction |
| url | http://dx.doi.org/10.1155/2014/645084 |
| work_keys_str_mv | AT thiagodossantosgomes differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction AT marianacoimbragarcia differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction AT flaviadesouzacunha differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction AT heloisawerneckdemacedo differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction AT josemauroperalta differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction AT reginahelenasaramagoperalta differentialdiagnosisofentamoebasppinclinicalstoolsamplesusingsybrgreenrealtimepolymerasechainreaction |