Comparación de dos protocolos de extracción de ADN para la detección de infección por Plasmodium spp. en Anopheles spp.

María Monsalve Restrepo; Héctor Ortega Arellano; Lina Gutiérrez Builes; Margarita M. Correa; Mario Zapata Tamayo

doi:10.17533/udea.hm.10877

Authors

María Monsalve Restrepo University of Antioquia
Héctor Ortega Arellano University of Antioquia
Lina Gutiérrez Builes University of Antioquia
Margarita M. Correa University of Antioquia
Mario Zapata Tamayo University of Antioquia

DOI:

https://doi.org/10.17533/udea.hm.10877

Keywords:

malaria, Anopheles spp., Plasmodium spp., DNA extraction, nested PCR

Abstract

Introduction

Detection of anophelines infected with Plasmodium spp. has traditionally been conducted by optical microscopy and immuno-assays. Currently, methodologies based on Polymerase Chain Reaction (PCR), provide high sensitivity and specificity; however, some authors have reported that inhibitors present in the mosquito’s body can decrease PCR sensitivity for Plasmodium spp. detection and that their effect could be reduced using DNA extraction protocols that efficiently remove those potential inhibitors.

ObJective

To compare two DNA extraction protocols for the detection of Plasmodium spp. infecting Anopheles spp.

Materials and methods

Ten Anopheles stephensi mosquitoes infected with Plasmodium falciparum were dissected to separate head-thorax and abdomen and processed by one of the extraction protocols, dilutions of the DNA were evaluated by nested PCR. Differences between amplification frequencies were analyzed by the t-Student’s test and the hypotheses were analyzed by the test of difference between proportions of two populations.

Results

In specimens with DNA extracted by the protocol without chelating resin amplification of parasite DNA was achieved in 11.6% of the samples and with the protocol with chelating resin, in 6.6%. From all of the amplifications, 20% corresponded to undiluted DNA and 7.5% to DNA diluted 1:10; the difference between amplification proportions was statistically significant (p <0.05).

Conclusions

The protocol that more efficiently detected parasite DNA in mosquitoes was the one without chelating resin; with it, amplification was approximately two fold to the one obtained with the protocol with resin.

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Author Biographies

María Monsalve Restrepo, University of Antioquia

Microbiologist and Bioanalyst, Molecular Microbiology Group, School of Microbiology, University of Antioquia. Medellín-Colombia.

Héctor Ortega Arellano, University of Antioquia

Microbiologist and Bioanalyst, Molecular Microbiology Group, School of Microbiology, University of Antioquia. Medellín- Colombia.

Lina Gutiérrez Builes, University of Antioquia

Microbiologist and Bioanalyst, Phd in Basic Biomedical Sciences, Molecular Microbiology Group, School of Microbiology. University of Antioquia, Medellin-Colombia.

Margarita M. Correa, University of Antioquia

Ph. D. in Microbiology, Professor, Molecular Microbiology Group, School of Microbiology, University of Antioquia. Medellín-Colombia.

Mario Zapata Tamayo, University of Antioquia

Bacteriologist and Bioanalyst, Epidemiologist and MSc. in Basic Biomedical Sciences, Veterinary Microbiology Group, School of Microbiology, University of Antioquia, Medellín- Colombia.

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