Title
Comparison of methods of dna extraction from giardia spp. Measured by conventional PCR
Other title
Comparación de métodos de extracción de adn de giardia spp. Medidos por PCR convencional
Date Issued
01 January 2019
Access level
open access
Resource Type
journal article
Publisher(s)
Instituto Nacional de Salud
Abstract
Objectives. To compare different methods of DNA extraction from cysts and trophozoites of Giardia spp. using the conventional polymerase chain reaction (PCR) technique. Materials and Methods. Cysts of Giardia spp. were isolated from 65 coprological samples from national reference hospitals, obtaining an average load of 5x104 parasites. In addition, Giardia intestinalis trophozoites (ATCC® 30957™) were cultured obtaining a 5x106 parasitic load. Eleven extraction methods for cysts and six for trophozoites were compared. The concentration and purity of the extracted DNA were determined by spectrophotometry and the extraction yield was assessed by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) genes with a semi nested PCR assay. Results. It was observed that method 1 showed the highest concentration of DNA from cysts (12.24 ng/µL), purity (1.4) and best performance (bg: 100% amplification; gdh: 60% amplification) compared to the other methods evaluated. In the case of trophozoites, the method without pre treatment showed the highest level of DNA concentration, purity, and yield (26.56 ng/µL; 1.85; 100% amplification of bg and gdh, respectively). Conclusions. Mechanical, thermal shock, and enzymatic pre-treatments are necessary for the rupture of the cystic wall of Giardia spp. making it the highest-yielding bg molecular marker for detecting cyst DNA. Trophozoites do not require pre-treatment to achieve satisfactory results. A reproducible methodology for the extraction of DNA from Giardia spp. from any evolutionary stage is available.
Start page
423
End page
432
Volume
36
Issue
3
Language
Spanish
OCDE Knowledge area
Gastroenterología, Hepatología Parasitología
Scopus EID
2-s2.0-85075240063
PubMed ID
Source
Revista Peruana de Medicina Experimental y Salud Publica
ISSN of the container
1726-4634
Sponsor(s)
Agradecemos a los laboratorios de parasitología de los hospitales nacionales y a los laboratorios de referencia nacional por el apoyo en la realización del presente proyecto. Fuentes de financiamiento: La presente investigaci?n fue financiada con recursos de Fondo Nacional de Ciencia y tecnología (FONDECYT), Convenio N° 136-2015 proyecto ?Caracterización molecular de Giardia spp. y su relación con el perfil de resistencia en hospitales de referencia nacional de Lima-Perú La aprobación ética para este estudio fue otorgada por el Comité Institucional de Ética en Investigación (CIEI) del Instituto Nacional de Salud de acuerdo con las normas éticas para la autorización de uso de material biológico para las investigaciones del Centro Nacional de Salud Pública.
Sources of information: Directorio de Producción Científica Scopus