Estandarización de una técnica molecular para el diagnóstico de dermatofitos
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Fecha
2009
Profesor/a Guía
Facultad/escuela
Idioma
es
Título de la revista
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Título del volumen
Editor
Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
Actualmente, el diagnóstico de dermatofitos en Chile se encuentra limitado sólo a las
técnicas de cultivo tradicionales, que demoran como mínimo 21 días para obtener un
resultado final. Molecularmente, el diagnóstico de dermatofitos en Chile llega sólo a la
presencia o ausencia de éstos. Del análisis realizado mediante cuatro métodos de
extracción, se obtuvo un 100% de resultados negativos al analizar muestras clínicas, no
así el caso de cepas, donde se obtuvo un 95,45% de positividad. Los partidores
Derma1 y Derma2, junto a los partidores dPsD1 mostraron 100% de negatividad en
muestras clínicas y cepas, contrario a lo observado con TR1 y TR2, los cuales
presentaron un 100% de positividad en cepas, no así en muestras clínicas.
Posiblemente los resultados negativos observados en muestras clínicas se deben a la
complejidad que presenta la ruptura celular de piel, pelo y uñas. En este estudio se
propone un protocolo para el diagnóstico de dermatofitos basado en un método de
preenriquecimiento, extracción y amplificación. Se observó que el punto clave del pre
enriquecimiento es el aporte de oxigeno a través de la agitación constante. De los 4
métodos de extracción analizados, sólo con el método de Tiocianato de
Guanidina/Silica (GTS) se obtuvieron resultados a partir de muestras clínicas, aunque
con baja reproducibilidad (4,3% ). Con respecto a los partidores utilizados, sólo con TR1
y TR2 se logró confirmar la especificidad y sensibilidad de la técnica previamente
estandarizada. Se comprobó que los partidores dPsD1 y Derma1 y 2, ambos diseñados
a partir de la secuencia de topoisomerasa II, no amplifican para secuencias de ADN en
cepas recolectadas a partir de muestras clínicas
Nawadays, dermataphyte diagnosis in Chile is limitad anly ta traditianal culture techniques, which take at least 21 days to produce end results. In Chile, molecular diagnosis of dermatophytosis only concludes the presence or absence of infection. In this study, tour extraction methods for clinical sample analysis produced 100% negativa results. On the other hand, using stocks produced 95.45% positive results. Employing Derma1 and Derma2 primers with dPsD1 primers showed 100% negativity in clinioal samples and stocks; on the contrary, making use of TR1 and TR2 showed 1 00% positiva results in stocks, but not in clinical samples. lt is possible that the negative results observed in clinical samples are related to the complexity of skin, hair and nail cellular breakdown. In this study, a pre.enrichment, extraction and amplification method based protocqf is proposed. Pre-enrichment key points are underlined. such as OXYQen supply through eonstant agitation. Only one of the four applied extraetion methods produced positive results: The Guanidinium Thiocyanate-Silica (GTS) one, but providing low reproducibility (4.3%) with clinical samples, in cantrast with stock-based analysis. In relation to the utilizad primers, confirmation of specificity and sensibility of the previously standardized technique was possible only with TR1 and TR2. lt was confirmed that primers dPsD1, Derma1 and Derma2 (synthesized fram a tapaisamerase 11 sequence) do not amplify for DNA sequences in stocks collected from clinical samples.
Nawadays, dermataphyte diagnosis in Chile is limitad anly ta traditianal culture techniques, which take at least 21 days to produce end results. In Chile, molecular diagnosis of dermatophytosis only concludes the presence or absence of infection. In this study, tour extraction methods for clinical sample analysis produced 100% negativa results. On the other hand, using stocks produced 95.45% positive results. Employing Derma1 and Derma2 primers with dPsD1 primers showed 100% negativity in clinioal samples and stocks; on the contrary, making use of TR1 and TR2 showed 1 00% positiva results in stocks, but not in clinical samples. lt is possible that the negative results observed in clinical samples are related to the complexity of skin, hair and nail cellular breakdown. In this study, a pre.enrichment, extraction and amplification method based protocqf is proposed. Pre-enrichment key points are underlined. such as OXYQen supply through eonstant agitation. Only one of the four applied extraetion methods produced positive results: The Guanidinium Thiocyanate-Silica (GTS) one, but providing low reproducibility (4.3%) with clinical samples, in cantrast with stock-based analysis. In relation to the utilizad primers, confirmation of specificity and sensibility of the previously standardized technique was possible only with TR1 and TR2. lt was confirmed that primers dPsD1, Derma1 and Derma2 (synthesized fram a tapaisamerase 11 sequence) do not amplify for DNA sequences in stocks collected from clinical samples.
Notas
Tesis (Tecnólogo Médico)
Palabras clave
Enfermedades de la Piel, Diagnóstico, Chile