Juan Felipe Ramirez, Alejandra De la Torre
Antecedentes. Los métodos convencionales para la identificación de levaduras del género Candida requieren mucho tiempo, necesitan cantidad importante de la muestra y, en muchos casos, es necesario realizar cultivo.Objetivos. Evaluar un par de cebadores para la identificación de levaduras del género Candida y demostrar su potencial aplicación para el diagnóstico de infecciones intraoculares.Materiales y métodos. Se utilizaron 17 cepas de referencia, 29 aislamientos clínicos de flujo vaginal, 7 muestras de humor acuoso y una de humor vítreo. Evaluamos tres métodos para el rompimiento de la pared celular: congelación descongelación, sonicación, y la enzima liticasa. Para la purificación del ácido nucleico, se utilizó en los tres casos el estuche comercial Wizard Genomics; en la reacción de PCR, el estuche comercial Go Taq Green Master Mix y los iniciadores ITS3 e ITS4 a una concentración de 0,5 ?M.
Background. Conventional methods for Candida identification are time consuming, require high amounts of the sample and in several cases the culture is mandatory.Aims. To evaluate two primers for the identification of Candida and to demonstrate their application for intraocular infections.Materials and methods. 17 Candida reference strains, 29 vaginal samples, 7 samples of aqueous humor and one of vitreous humor were studied. Three methods to lyse the Candida wall were analyzed: freezing-thawing, sonication and the use of lyticase. For nucleic acid purification the Wizard Genomics kit was used; in the PCR the commercial kit Go Taq Green Master Mix and the primers ITS3 and ITS4 were used.Results. The best method for lysis was the enzymatic lysis with lyticase. The primers ITS3 and ITS4 identified all the Candida strains and there were no cross-reaction with other microorganisms. The sensitivity of the PCR was 100 fg. All the samples of vaginal flux and Candida strains were identified and only one sample of aqueous humor was positive for the genus Candida. In the other samples of aqueous humor toxoplasmosis was the definitive diagnosis.Conclusion. PCR amplification for Candida is a sensitive and specific technique for the diagnosis.
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