PCR-RFLP and RAPD for typing neotropical Leishmania
Keywords:
Leishmania, leishmaniasis/diagnosis, polymerase chain reaction, polymorphism, restriction fragment length
Abstract
Introduction. The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification.Objectives. Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples.
Materials and methods. DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5.
Results. PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed.
Conclusions. The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.
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References
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11. Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: Current status and future applications. J Clin Microbiol. 2007;45:21-5.
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13. Berman JD. Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis. 1997;24:684-703.
14. Singh S, Sivakumar R. Recent advances in the diagnosis of leishmaniasis. J Postgrad Med. 2003; 49:55-60.
15. Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn. 2003;3:657-67.
16. Cupolillo E, Grimaldi G Jr, Momen H, Beverly SM. Intergenic region typing (IRT):a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol. 1995;73:145-55.
17. Bañuls AL, Jonquieres R, Guerrini F, LePont F, Barrera C, Espinel I, et al. Genetic analysis of Leishmania parasites in Ecuador: are Leishmania (Viannia) panamensis and Leishmania (V) guyanensis distinct taxa? Am J Trop Med Hyg. 1999;61:828-45.
18. Montalvo AM, Fraga J, Romero JA, Monzote L, Montano I, Dujardin JC. PCR-RFLP/HsP70 para identificar y tipificar Leishmania de la región neotropical. Rev Cub Med Trop. 2006;58(3). [Fecha de consulta: 24 de octubre de 2008]. Disponible en: http:// scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0375- 07602006000300009&lng=es&nrm=iso.
19. Bhattacharyya R, Singh R, Hazra TK, Majumder HK. Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites. FEMS Microbiol Lett. 1993;114:99-104.
20. Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarechy D, Ayala FJ. Genetic characterization of six parasitic protozoa: a parity between random-primer DNA typing and multilocus enzyme electrophoresis. Proc Natl Acad Sci USA. 1993;90:1335-9.
21. Diakou A, Dovas CI. Optimization of random amplified polymorphic DNA producing amplicons of 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates. Anal Biochem. 2001;288:195-200.
22. Hanafi R, Barhoumi M, Ali SB, Guizani I. Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species complex. Exp Parasitol. 2001;98:90-9. 23. Zemanova E, Jirku M, Mauruicio IL, Miles MA, Lukes J. Genetic polymorphism within the Leishmania donovani complex: correlation with geographic origin. Am J Trop Med Hyg. 2004;70:613-7.
24. Martínez E, Alonso V, Quispe A, Thomas MC, Alonso R, Pinero JE, et al. RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis. Parasitology. 2003;127:513-7.
25- Rodríguez-Bonfante C, Bonfante-Garrido R, Grimaldi G Jr, Momen H, Cupolillo E. Genotypically distinct Leishmania colombiensis isolates from Venezuela cause both cutaneous and visceral Leishmaniasis in humans. Infect Genet Evol. 2003;3:119-24.
26- García L, Kindt A, Bermúdez H, Llanos-Cuentas A, De Doncker S, Arévalo J, et al. Culture- independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004;42:2294-7.
2. World Health Organization. Tropical disease research. Progress 1975-94, highlights 1993-94. Twelfth Programme Report of the UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Disease (TDR). Geneva: WHO; 1995. p.135-46.
3. Rotureau B, Ravel C, Couppie P, Pratlong F, Nacher M, Dedet JP, et al. Use of PCR-restriction fragment length polymorphism analysis to identify the main New World Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol. 2006;44:459-67.
4. Victoir K, De Doncker S, Cabrera L, Álvarez E, Arévalo J, Llanos-Cuentas A, et al. Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg. 2003;97:80-7.
5. Serin MS, Daglioglu K, Bagirova M, Allahverdiyev A, Uzun S, Vural Z, et al. Rapid diagnosis and genotyping of Leishmania isolates from cutaneous and visceral leishmaniasis by microcapillary cultivation and polymerase chain reaction-restriction fragment length polymorphism of miniexon region. Diagn Microbiol Infect Dis. 2005; 53:209-14.
6. Noyes HA, Belli AA, Maingon R. Appraisal of various random amplified polymorphic DNA polymerase Chain reaction primers for Leishmania identification. Am J Trop Med Hyg. 1996;55:98-105.
7. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. Second edition. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1989.
8. Ordeñana-Pilotos R. Optimización de la técnica del ADN polimórfico amplificado al azar (RAPD), para la identificación de especies de Leishmania (tesis). Ciudad de La Habana: Instituto Pedro Kourí; 2005.
9. Sneath PH, Sokal RR. Numerical taxonomy. San Francisco: Freeman WH & Co.; 1973.
10. Pavlícèk A, Hrdá S, Flegr J. Free tree-freeware program for construction of phylogenetic trees on the basis of distance data and for bootstrap/jackknife analysis of the threes robustness. Application in the RAPD analysis of genus Frenkelina. Folia Biol (Praha). 1999;45:97-9.
11. Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: Current status and future applications. J Clin Microbiol. 2007;45:21-5.
12. Croft SL, Yardley V, Kendrick H. Drug sensitivity of Leishmania species: some unresolved problems. Trans R Soc Trop Med Hyg. 2002;96(Suppl.1):127-9.
13. Berman JD. Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis. 1997;24:684-703.
14. Singh S, Sivakumar R. Recent advances in the diagnosis of leishmaniasis. J Postgrad Med. 2003; 49:55-60.
15. Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn. 2003;3:657-67.
16. Cupolillo E, Grimaldi G Jr, Momen H, Beverly SM. Intergenic region typing (IRT):a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol. 1995;73:145-55.
17. Bañuls AL, Jonquieres R, Guerrini F, LePont F, Barrera C, Espinel I, et al. Genetic analysis of Leishmania parasites in Ecuador: are Leishmania (Viannia) panamensis and Leishmania (V) guyanensis distinct taxa? Am J Trop Med Hyg. 1999;61:828-45.
18. Montalvo AM, Fraga J, Romero JA, Monzote L, Montano I, Dujardin JC. PCR-RFLP/HsP70 para identificar y tipificar Leishmania de la región neotropical. Rev Cub Med Trop. 2006;58(3). [Fecha de consulta: 24 de octubre de 2008]. Disponible en: http:// scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0375- 07602006000300009&lng=es&nrm=iso.
19. Bhattacharyya R, Singh R, Hazra TK, Majumder HK. Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites. FEMS Microbiol Lett. 1993;114:99-104.
20. Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarechy D, Ayala FJ. Genetic characterization of six parasitic protozoa: a parity between random-primer DNA typing and multilocus enzyme electrophoresis. Proc Natl Acad Sci USA. 1993;90:1335-9.
21. Diakou A, Dovas CI. Optimization of random amplified polymorphic DNA producing amplicons of 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates. Anal Biochem. 2001;288:195-200.
22. Hanafi R, Barhoumi M, Ali SB, Guizani I. Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species complex. Exp Parasitol. 2001;98:90-9. 23. Zemanova E, Jirku M, Mauruicio IL, Miles MA, Lukes J. Genetic polymorphism within the Leishmania donovani complex: correlation with geographic origin. Am J Trop Med Hyg. 2004;70:613-7.
24. Martínez E, Alonso V, Quispe A, Thomas MC, Alonso R, Pinero JE, et al. RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis. Parasitology. 2003;127:513-7.
25- Rodríguez-Bonfante C, Bonfante-Garrido R, Grimaldi G Jr, Momen H, Cupolillo E. Genotypically distinct Leishmania colombiensis isolates from Venezuela cause both cutaneous and visceral Leishmaniasis in humans. Infect Genet Evol. 2003;3:119-24.
26- García L, Kindt A, Bermúdez H, Llanos-Cuentas A, De Doncker S, Arévalo J, et al. Culture- independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004;42:2294-7.
How to Cite
1.
Montalvo AM, Monzote L, Fraga J, Montano I, Muskus C, Marín M, et al. PCR-RFLP and RAPD for typing neotropical Leishmania. Biomed. [Internet]. 2008 Dec. 1 [cited 2025 Apr. 4];28(4):597-606. Available from: https://revistabiomedicaorg.biteca.online/index.php/biomedica/article/view/66
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