Hostname: page-component-77c89778f8-vsgnj Total loading time: 0 Render date: 2024-07-18T18:54:39.890Z Has data issue: false hasContentIssue false
  • English
  • Français

An alternative method for DNA extraction and PCR identification of Entamoeba histolytica and E. dispar in fecal samples

Published online by Cambridge University Press:  02 June 2009

E. N. VIANNA ,
J. O. COSTA ,
C. K. S. SANTOS ,
M. C. CURY ,
E. F. SILVA ,
A. O. COSTA  and
M. A. GOMES
E. N. VIANNA
Affiliation:
Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627-31270-901, Belo Horizonte, Minas Gerais, Brazil
J. O. COSTA
Affiliation:
Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627-31270-901, Belo Horizonte, Minas Gerais, Brazil
C. K. S. SANTOS
Affiliation:
Laboratório de Parasitologia, ICB, Universidade Federal de Uberlândia, Minas Gerais, Brazil
M. C. CURY
Affiliation:
Laboratório de Parasitologia, ICB, Universidade Federal de Uberlândia, Minas Gerais, Brazil
E. F. SILVA
Affiliation:
Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627-31270-901, Belo Horizonte, Minas Gerais, Brazil
A. O. COSTA
Affiliation:
Departamento de Patologia Básica, SCB, Universidade Federal do Paraná, Curitiba, Paraná, Brazil
M. A. GOMES*
Affiliation:
Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627-31270-901, Belo Horizonte, Minas Gerais, Brazil
*
*Corresponding author: Departamento de Parasitologia, ICB-UFMG, Av. Antonio Carlos, 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil. Tel: +55 31 34092846. Fax: +55 31 34092970. E-mail: magomes@icb.ufmg.br
Get access Rights & Permissions [Opens in a new window]

Summary

Since it is known that Entamoeba dispar is non-pathogenic and morphologically similar to E. histolytica, there are many targets used in PCR for differentiating these species. However, obtaining high quality DNA from fecal samples is fundamental for PCR. Most methods are laborious or use kits that make diagnosis expensive. In the present work, a new simple, fast and cheap technique of DNA extraction from fecal samples was combined with a PCR for an episomal target in order to identify E. histolytica and E. dispar in feces.

Keywords

Entamoeba histolyticaEntamoeba dispardifferential diagnostic
Type
Research Article
Information
Parasitology , Volume 136 , Issue 7 , June 2009 , pp. 765 - 769
Copyright
Copyright © Cambridge University Press 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Anderson, K. L. and Lebepe-Mazur, S. (2003) Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig. Journal of Applied Microbiology 94, 988–993.CrossRefGoogle ScholarPubMed
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Truhl, K. (1994). Section I – Preparation of genomic DNA-phenol extraction and ethanol precipitation of DNA-unit 2.1. In Currents Protocols in Molecular Biology. Massachusetts General Hospital and Harvard Medical School, USA.Google Scholar
Barral de Martinez, A. M., Gomes, M. A., Viana, J. C., Romanha, A. J. and Silva, E. F. (1996). Isoenzime profile as parameter to differentiate pathogenic strains of Entamoeba histolytica in Brazil. Revista do Instituto de Medicina Tropical de São Paulo 38, 407–412.Google Scholar
Bhattacharya, S., Som, I. and Bhattacharya, A. (1998). The ribosomal DNA plasmids of Entamoeba. Parasitology Today 14, 181–185. doi:10.1016/S0169-4758(98)01222-8.CrossRefGoogle ScholarPubMed
Blessmann, J., Buss, H., Nu, P. A., Dinh, B. T., Ngo, Q. T., Van, A. L., Abd-Alla, M. D., Jackson, T. F. H. G., Ravdin, J. I. and Tannich, E. (2002) Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. Journal of Clinical Microbiology 40, 4413–4417. doi:10.1128/jcm.40.12.4413-4417.2002.CrossRefGoogle ScholarPubMed
Diamond, L. S. and Clark, C. G. (1993). A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. The Journal of Eukaryotic Microbiology 40, 340–344.CrossRefGoogle ScholarPubMed
Gomes, M. A., Pesquero, J. B., Furst, C., Valle, P. R., Pesquero, J. L. and Silva, E. F. (1999). An improved method to distinguish Entamoeba histolytica and Entamoeba dispar. Parasitology 119, 359–362.CrossRefGoogle ScholarPubMed
Haque, R., Mollah, N. U., Ali, I. K. M., Alam, K., Eubanks, A., Lyerly, D. and Petri, W. A. Jr. (2000). Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. Journal of Clinical Microbiology 38, 3235–3239.CrossRefGoogle ScholarPubMed
Hoffman, W. A., Pons, J. A. and Janer, J. L. (1934). The sedimentation concentration method in Schistosomiasis mansoni. Journal of Public Health 9, 281–298.Google Scholar
Khairnar, K. and Parija, S. C. (2007). A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar in DNA in stool samples. BMC Microbiology 7, 47–55. doi: 10.1186/1471-2180-7-47.CrossRefGoogle Scholar
Mirelman, D., Nuchamowitz, Y. and Stolarsk, T. (1997). Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. Journal of Clinical Microbiology 35, 2405–2407.CrossRefGoogle ScholarPubMed
Paglia, M. G. and Visca, P. (2004). An improved PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in formalin-fixed stools. Acta Tropica 92, 273–277. doi: 10.1016/j.actatropica.2004.09.003.CrossRefGoogle ScholarPubMed
Petri, W. A. Jr., Haque, R., Lyerly, D. and Vines, R. R. (2000). Estimating the impact of amebiasis on health. Parasitology Today 16, 320–321.CrossRefGoogle ScholarPubMed
Qvarnstrom, Y., James, C., Xayavong, M., Holloway, B. P., Visvesvara, G. S., Sriram, R. and Silva, A. J. (2005). Comparison of real time PCR protocols for differential laboratory diagnosis of amebiasis. Journal of Clinical Microbiology 43, 5491–5497. doi: 10.1128/jcm43.11.5491-5497-2005.CrossRefGoogle ScholarPubMed
Ramos, F., Zurabian, R., Morán, P., Ramiro, M., Gómez, A., Clark, C. G., Melendro, E. I., García, G. and Ximénez, C. (1999). The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica. Transactions of Royal Society of Tropical Medicine and Hygiene 93, 335–336.CrossRefGoogle ScholarPubMed
Roy, S., Kabir, M., Mondal, D., Ali, I. B. K. M., Petri, W. A. Jr. and Haque, R. (2005). Real time – PCR Assay for diagnosis of Entamoeba histolytica infection. Journal of Clinical Microbiology 43, 2168–2172. doi: 10.1128/jcm.43.5.2168-2172.2005.CrossRefGoogle ScholarPubMed
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Small scale preparation of plasmid DNA- Lysis by alkali. In Molecular Cloning: A Laboratory Manual (ed. Nolan, C.), pp. 1.25–1.31. Cold Spring Harbor Laboratory Press, New York, USA.Google Scholar
Tannich, E., Bruchhaus, I., Walter, R. D. and Horstmann, R. D. (1991). Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase. Molecular and Biochemistry Parasitology 49, 61–72.CrossRefGoogle ScholarPubMed
Tanyuksel, M. and Petri, W. A. Jr. (2003). Laboratory diagnosis of amebiasis. C linical Microbiology Reviews 16, 713–729. doi: 10.1128/cmr.16.4.713-729.2003.CrossRefGoogle ScholarPubMed
Troll, H., Marti, H. and Weiss, N. (1997). Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. Journal of Clinical Microbiology 35, 1701–1705.Google Scholar
World Health Organization (1997). Expert consultation on amoebiasis: Amoebiasis. Weekly Epidemiological Record 72, 97–100.Google Scholar
Young, K. H., Bullock, S. L., Melvin, D. M. and Spruill, C. L. (1979). Ethyl acetate as a substitute for diethyl ether in the formalin-ether sedimentation technique. Journal of Clinical Microbiology 10, 852–853.CrossRefGoogle ScholarPubMed
Zengzhu, G., Bracha, R., Nuchamowitz, Y., Cheng-I, W. and Mirelman, D. (1999). Analysis by enzyme-linked immunosorbent assay and PCR of human liver abscess aspirates from patients in China for Entamoeba histolytica. Journal of Clinical Microbiology 37, 3034–3036.Google Scholar