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Seminario molecular

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Pablo Andres Lara
ID: 000133477
3er semestre - Medicina UPB
2010

Published in: Health & Medicine
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Seminario molecular

  1. 1. Highly sensitive non-isotopic restrictionendonucleaseFIngerprinting of nucleotidevariability in the gp60 gene withinCryptosporidium species, genotypes andsubgenotypes infective to humans, and itsimplications<br />AradhanaPangasa<br />Department of VeterinaryScience, TheUniversity of Melbourne, Werribee, Australia<br />Pablo Andrés Lara<br />3er Semestre – Medicina UPB<br />
  2. 2. INTRODUCTION<br />Geneticvariationsanalysis leads toidentification of microorganisms’sspecies and subspecies<br />Geneticvariations leads toanadequateclassification of microorganisms<br />Diverseamplification and separationtechniques are used<br />
  3. 3. Cryptosporidium<br />Protozoan parasite<br />Apicomplexa<br />No vectors<br />Humanfaeces<br />Mostcommonspecies in humans are C. Parvumand C. hominis<br />
  4. 4. gp60 gene<br />Codifies 15 and 40 kDacell-surfaceglycoproteinscalled GP15 and GP40 respectively<br />This genes are involved in invasion of host cellbyCrypstosporidium<br />
  5. 5. HumanInfection<br />Known as Cryptosporidiosis<br />Affectsdigestivesystem<br />Usuallyacuteepisodes<br />Causes diarrhea<br />Commoninfection in AIDS patients<br />
  6. 6. General Objective<br />Demostratetheefectiveness of using PCR-basedrestrictionanalysisto determine and classifyseveralspecies of Cryptosporidium. Thisclasificationwas done detectingseveralmutations in the gp60 gene found in themicroorganism<br />
  7. 7. Métodos y Materiales<br />Obtención de muestras (heces contaminadas humanas y de rumiantes)<br />Tinción (Ziehl – Neelsen)<br />Heces suspendidas en buffer de fosfato (pH=7.4)<br />
  8. 8. Métodos y Materiales<br />Muestra llevada a 100°C y luego centrifugada<br />ADN genómico obtenido con kit MoBio<br />
  9. 9. Reacción en cadena de la polimerasa<br />Sirve para la amplificacion de material genético<br />Incremento geométrico de una secuencia<br />De poco ADN, se puede obtener mucho<br />En este caso, sirve para multiplicar la secuencia que codifica los gp60, y formar amplicons<br />
  10. 10. Reacción en cadena de la Polimerasa<br />Los amplicons luego son sometidos a REF, que son endonucleasas de restricción.<br />Por enzimas de restricción, y luego electroforesis, puedo saber cada cepa tiene cual variación del gen<br />Esto lo logra cortando secuencias especificas de nucleotidos<br />
  11. 11. Resultados<br />
  12. 12. Resultados<br />
  13. 13. Resultados<br />Por electroforesis se encontraron 13 cepas de Hominisy 14 cepas de Parvum<br />Se detectó el cambio de UN par de bases, demostrando alta detección (Una T por G)<br />Hay menos variación de nucleótidos de gp60 en Hominisque en Parvum<br />
  14. 14. Discussion<br />
  15. 15. Discussion<br />
  16. 16. Discussion<br />
  17. 17. Conclussions<br />The use of PCR plus REF leads togreatknowledge of differentCryptosporidiumspecies.<br />Theknowledge of differentspeciesmeansbettertreatment and epidemiologicalsurveilance<br />
  18. 18. Conclussions<br />REF ishighly detective of even single nucleotidealterations, in bothspecies<br />Otherprotozoansspecies can beclassifiedproperly (taxonomy) usingthistechnique<br />

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