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Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati

Published online by Cambridge University Press:  17 February 2021

Héctor Gabriel Avila Open the ORCID record for Héctor Gabriel Avila [Opens in a new window] ,
Marikena Guadalupe Risso ,
Paula Ruybal ,
Silvia Analía Repetto ,
Marcos Javier Butti ,
Marcos David Trangoni ,
Sylvia Grune Löffler ,
Verónica Mirtha Pérez  and
María Victoria Periago
Héctor Gabriel Avila*
Affiliation:
Laboratorio Provincial de Zoonosis de San Juan, Facultad de Ciencias Veterinarias, Facultad de Ciencias Químicas y Tecnológicas, Universidad Católica de Cuyo, San Luis, San Juan, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas, CABA, Argentina
Marikena Guadalupe Risso
Affiliation:
Instituto de Investigaciones en Microbiología y Parasitología Médica, IMPAM-UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, CABA, Argentina
Paula Ruybal
Affiliation:
Instituto de Investigaciones en Microbiología y Parasitología Médica, IMPAM-UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, CABA, Argentina
Silvia Analía Repetto
Affiliation:
Instituto de Investigaciones en Microbiología y Parasitología Médica, IMPAM-UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, CABA, Argentina
Marcos Javier Butti
Affiliation:
Laboratorio de Parasitosis Humanas y Zoonosis Parasitarias, Cátedra de Parasitología Comparada, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Buenos Aires, Argentina
Marcos David Trangoni
Affiliation:
Laboratorio de Brucella, Campylobacter y Microbiota del rumen, Instituto de Biotecnología/Instituto de Agrobiotecnología y Biología Molecular (IB/IABIMO), UEDD INTA-CONICET, CICVyA, CNIA, INTA Castelar, Hurlingham, Buenos Aires, Argentina
Sylvia Grune Löffler
Affiliation:
Laboratorio de Leptospirosis, Instituto de Patobiología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Instituto Nacional de Tecnología Agropecuaria Castelar, Buenos Aires, Argentina
Verónica Mirtha Pérez
Affiliation:
Sección de Rabia y Zoonosis, Dirección de Epidemiología, Ministerio de Salud Pública de San Juan, San Juan, Argentina
María Victoria Periago
Affiliation:
Consejo Nacional de Investigaciones Científicas y Técnicas, CABA, Argentina Fundación Mundo Sano, Buenos Aires, Argentina
*
Author for correspondence: Héctor Gabriel Avila, E-mail: [email protected]
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Abstract

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15–120 min), and different concentrations of malachite green dye (0.004–0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10–100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

Keywords

Copro-diagnosisLoop-Mediated Isothermal AmplificationLow-costToxocara canisToxocara catiToxocariasis
Type
Research Article
Information
Parasitology , Volume 148 , Issue 7 , June 2021 , pp. 819 - 826
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Copyright © The Author(s) 2021. Published by Cambridge University Press

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