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Comparison of different PCR amplification targets for molecular diagnosis of Strongyloides stercoralis

Published online by Cambridge University Press:  17 November 2023

F. Marquet ,
N. Mora ,
R.N. Incani ,
J. Jesus ,
N. Méndez ,
R. Mujica ,
H. Trosel  and
E. Ferrer Open the ORCID record for E. Ferrer [Opens in a new window]
F. Marquet
Affiliation:
Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED). Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela
N. Mora
Affiliation:
Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED). Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela
R.N. Incani
Affiliation:
Departamento de Parasitología, Facultad de Ciencias de la Salud Sede Carabobo, Universidad de Carabobo, Valencia, estado Carabobo, Venezuela
J. Jesus
Affiliation:
Departamento de Parasitología, Facultad de Ciencias de la Salud Sede Carabobo, Universidad de Carabobo, Valencia, estado Carabobo, Venezuela
N. Méndez
Affiliation:
Departamento de Parasitología, Facultad de Ciencias de la Salud Sede Carabobo, Universidad de Carabobo, Valencia, estado Carabobo, Venezuela
R. Mujica
Affiliation:
Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED). Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela
H. Trosel
Affiliation:
Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED). Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela
E. Ferrer*
Affiliation:
Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED). Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela Departamento de Parasitología, Facultad de Ciencias de la Salud Sede Aragua, Universidad de Carabobo, Maracay, estado Aragua, Venezuela
*
Corresponding author: E. Ferrer; Email: elizabeth.ferrer@gmail.com
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Abstract

Molecular techniques are an alternative for the diagnosis of strongyloidiasis, produced by Strongyloides stercoralis. However, it is necessary to determine the best amplification target for the populations of this parasite present in a geographical area and standardize a polymerase chain reaction (PCR) protocol for its detection. The objectives of this work were the comparison of different PCR targets for molecular detection of S. stercoralis and the standardization of a PCR protocol for the selected target with the best diagnostic results. DNA extraction was performed from parasite larvae by saline precipitation. Three amplification targets of the genes encoding ribosomal RNA 18S (18S rDNA) and 5.8S (5.8S rDNA) and cytochrome oxidase 1 (COX1) of S. stercoralis were compared, and the PCR reaction conditions for the best target were standardized (concentration of reagents and template DNA, hybridization temperature, and number of cycles). The analytical sensitivity and specificity of the technique were determined. DNA extraction by saline precipitation made it possible to obtain DNA of high purity and integrity. The ideal target was the 5.8S rDNA, since the 18S rDNA yielded non-reproducible results and COX1 never amplified under any condition tested. The optimal conditions for the 5.8S rDNA-PCR were: 1.5 mM MgCl2, 100 μM dNTPs, 0.4 μM primers, and 0.75 U DNA polymerase, using 35 cycles and a hybridization temperature of 60 °C. The analytical sensitivity of the PCR was 1 attogram of DNA, and the specificity was 100%. Consequently, the 5.8S rDNA was shown to be highly sensitive and specific for the detection of S. stercoralis DNA.

Keywords

Strongyloides stercoralis5.8SstandardizationPCRdiagnosis
Type
Short Communication
Information
Journal of Helminthology , Volume 97 , 2023 , e88
Copyright
© The Author(s), 2023. Published by Cambridge University Press

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