ABSTRACT
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Subject(s)
Cell Fractionation/methods , Cell Separation/methods , Feces/parasitology , Giardia/isolation & purification , Oocysts , Oocysts/isolation & purification , Animals , DNA, Protozoan/isolation & purification , Dogs , Electrophoresis, Agar Gel , Endopeptidase K/pharmacology , Giardia/cytology , Giardia/genetics , Hot Temperature , Humans , Oocysts/chemistry , Oocysts/drug effects , Osmotic Pressure , Sodium Chloride/pharmacology , Solutions , Stress, Mechanical , Sucrose/pharmacologyABSTRACT
El objetivo de este trabajo fue optimizar y evaluar las técnicas de purificación, aislamiento y ruptura de quistes de Giardia spp a partir de heces formoladas para la obtención de ADN. La materia fecal filtrada fue sometida a 3 técnicas de purificación, utilizando soluciones de formol-éter, sacarosa y formol-éter más sacarosa. La solución de sacarosa permitió aislar los quistes con menos detritos. Los quistes purificados fueron tratados con 3 técnicas para la ruptura de los mismos: shock osmótico y calor, degradación química y shock térmico, acción enzimática y efecto mecánico. Solamente con la técnica de shock térmico, acción enzimática y efecto mecánico se observaron bandas fluorescentes en geles de agarosa. Los resultados de este trabajo permiten contar con una metodología de rutina, simple, que podría ser usada en los pasos previos a la técnica de PCR para la genotipificación de este parásito.
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Subject(s)
Animals , Dogs , Humans , Cell Fractionation/methods , Cell Separation/methods , Feces/parasitology , Giardia/isolation & purification , Oocysts , Oocysts/isolation & purification , DNA, Protozoan/isolation & purification , Electrophoresis, Agar Gel , Endopeptidase K/pharmacology , Giardia/cytology , Giardia/genetics , Hot Temperature , Osmotic Pressure , Oocysts/chemistry , Oocysts/drug effects , Solutions , Stress, Mechanical , Sodium Chloride/pharmacology , Sucrose/pharmacologyABSTRACT
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
