DETECCIÓN DIFERENCIAL DE TRYPANOSOMA EVANSI Y TRYPANOSOMA VIVAX MEDIANTE UN ENSAYO DE REACCIÓN EN CADENA DE LA POLIMERASA
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El propósito de este trabajo fue optimizar un ensayo de reacción en cadena de la polimerasa (PCR) para efectuar el diagnóstico diferencial de Trypanosoma evansi yTrypanosoma vivax, usando cebadores previamente descritos: 21-mer/22-mer e ILO1264/ILO1265. La especificidad se evaluó efectuando pruebas preliminares sobre muestras de ADN purificado de diversas especies de parásitos y sobre mezclas de éstas. La sensibilidad se valoró empleando diluciones de ADN deaislados de referencia de T. evansi y T. vivax y concentraciones variables de cebadores. El ensayo optimizado mostró una sensibilidad de 10 pg de ADN, con especificidad para el Subgénero Trypanozoon (cebadores 21-mer/22-mer) y especificidad absoluta para T. vivax (cebadores ILO1264/ILO1265). Ambos grupos de cebadores mostraron potencialidad para detectar infecciones mixtas T. evansi/T. vivax. Un análisis de hibridación sobre el patrón de cariotipo de diversos Kinetoplastida, usando la sonda Te- ADN, mostró su carácter repetitivo y la distribución de su secuencia en diferentes cromosomas de T. evansi TE0. La PCR fue evaluada como herramienta diagnóstico de la presencia de tripanosomas en muestras sanguíneas de animales con infecciones experimentales, demostrando alta sensibilidad al detectar positividad hasta 72 horas antes que lo hicieran los métodos parasitológicos. Este ensayo también fue evaluado sobre muestras sanguíneas de búfalos de agua con infecciones naturales, mostrando alta sensibilidad y especificidad al detectar 9/86 muestras como positivas a T. vivax, revelando una tasa de infección activa de 10,47%; valor tres veces superior a los resultados de la evaluación microscópica de frotis teñidos (3,49%) y casi dos veces superior (6,98%) a la técnica parasitológica de microcentrifugación capilar. En ninguna de las muestras de búfalos evaluadas se detectó T. evansi.
Differential Detection of Trypanosoma evansi and Trypanosoma vivax Through a Polymerase Chain Reaction Assay
ABSTRACT
The purpose of this study was to standardize a polymerase chain reaction (PCR) assay in order to reach a differential diagnosis of Trypanosoma evansi and Trypanosoma vivax using previously described primers: 21-mer/22-mer and ILO1264/ILO1265. Assay specificity was evaluated through preliminary test on both purified DNA from different parasite species and on their mixtures. Assay sensitivity was tested using decreasing DNA concentration of T. evansi and T. vivax strain reference and different primer concentrations. The optimized PCR assay showed a sensitivity of 10 pg of DNA with specificity to subgenus Trypanozoon (primers 21-mer/22-mer) and an absolute specificity for T. vivax (ILO1264/ILO1265). Both primer sets showed a high capability to detect mixed infections by T. evansi/T. vivax. A hybridization assay on molecular karyotypes of Kinetoplastida parasites, using the Te-DNA probe, showed both a high sequence copying and a sequence distribution in different chromosomes of T. evansi stock TE0. PCR assay was used as a tool to evaluate the presence of tr ypanosomes in blood samples of experimentally infected animals, showing high sensitivity as it detected positive samples 72 hours before than parasitological methods. This assay was also run on blood samples of naturally infected water buffaloes showing both high sensitivity and specificity as it detected 9 out of 86 samples as positive for T. vivax. This reveals an active infection range of 10.47%. A value which is three times higher than the detection range by microscopic evaluation of stained blood smears (3.49%) and about twice as high as the microhematocrit centrifugation technique (6.98%). T. evansi was not found in any of the buffalo blood samples tested.
Differential Detection of Trypanosoma evansi and Trypanosoma vivax Through a Polymerase Chain Reaction Assay
ABSTRACT
The purpose of this study was to standardize a polymerase chain reaction (PCR) assay in order to reach a differential diagnosis of Trypanosoma evansi and Trypanosoma vivax using previously described primers: 21-mer/22-mer and ILO1264/ILO1265. Assay specificity was evaluated through preliminary test on both purified DNA from different parasite species and on their mixtures. Assay sensitivity was tested using decreasing DNA concentration of T. evansi and T. vivax strain reference and different primer concentrations. The optimized PCR assay showed a sensitivity of 10 pg of DNA with specificity to subgenus Trypanozoon (primers 21-mer/22-mer) and an absolute specificity for T. vivax (ILO1264/ILO1265). Both primer sets showed a high capability to detect mixed infections by T. evansi/T. vivax. A hybridization assay on molecular karyotypes of Kinetoplastida parasites, using the Te-DNA probe, showed both a high sequence copying and a sequence distribution in different chromosomes of T. evansi stock TE0. PCR assay was used as a tool to evaluate the presence of tr ypanosomes in blood samples of experimentally infected animals, showing high sensitivity as it detected positive samples 72 hours before than parasitological methods. This assay was also run on blood samples of naturally infected water buffaloes showing both high sensitivity and specificity as it detected 9 out of 86 samples as positive for T. vivax. This reveals an active infection range of 10.47%. A value which is three times higher than the detection range by microscopic evaluation of stained blood smears (3.49%) and about twice as high as the microhematocrit centrifugation technique (6.98%). T. evansi was not found in any of the buffalo blood samples tested.
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