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Seminario molecular

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Pablo Andres Lara

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

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Seminario molecular Seminario molecular Presentation Transcript

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