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Volume 13
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Article

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens

by
Martha V. Fernandez-Baca
1,†,
Alejandro Castellanos-Gonzalez
2,†,
Rodrigo A. Ore
1,
Jose L. Alccacontor-Munoz
1,
Cristian Hoban
3,
Carol A. Castro
1,
Melinda B. Tanabe
2,
Maria L. Morales
1,4,
Pedro Ortiz
3,
A. Clinton White, Jr.
2,4,
Miguel M. Cabada
1,2,4,* and
on behalf of the Fasciola TMRC in Peru
‡
1
Sede Cusco—Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Cusco 08002, Peru
2
Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA
3
Facultad de Ciencias Veterinarias, Universidad Nacional de Cajamarca, Cajamarca 06003, Peru
4
Universidad Peruana Cayetano Heredia-University of Texas Medical Branch Collaborative Research Center, Universidad Peruana Cayetano Heredia, Cusco 08002, Peru
*
Author to whom correspondence should be addressed.
†
These authors contributed equal to this work.
‡
A list of the Fasciola TMRC in Peru investigators is provided in the acknowledgement section of the manuscript.
Pathogens 2024, 13(6), 440; https://doi.org/10.3390/pathogens13060440
Submission received: 30 April 2024 / Revised: 16 May 2024 / Accepted: 21 May 2024 / Published: 23 May 2024
(This article belongs to the Special Issue Parasite Infection and Tropical Infectious Diseases)
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Abstract

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.
Keywords: molecular diagnostics; Fasciola hepatica; mini-PCR; RT-PCR; ITS-1 gene molecular diagnostics; Fasciola hepatica; mini-PCR; RT-PCR; ITS-1 gene

Share and Cite

MDPI and ACS Style

Fernandez-Baca, M.V.; Castellanos-Gonzalez, A.; Ore, R.A.; Alccacontor-Munoz, J.L.; Hoban, C.; Castro, C.A.; Tanabe, M.B.; Morales, M.L.; Ortiz, P.; White, A.C., Jr.; et al. A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens. Pathogens 2024, 13, 440. https://doi.org/10.3390/pathogens13060440

AMA Style

Fernandez-Baca MV, Castellanos-Gonzalez A, Ore RA, Alccacontor-Munoz JL, Hoban C, Castro CA, Tanabe MB, Morales ML, Ortiz P, White AC Jr., et al. A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens. Pathogens. 2024; 13(6):440. https://doi.org/10.3390/pathogens13060440

Chicago/Turabian Style

Fernandez-Baca, Martha V., Alejandro Castellanos-Gonzalez, Rodrigo A. Ore, Jose L. Alccacontor-Munoz, Cristian Hoban, Carol A. Castro, Melinda B. Tanabe, Maria L. Morales, Pedro Ortiz, A. Clinton White, Jr., and et al. 2024. "A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens" Pathogens 13, no. 6: 440. https://doi.org/10.3390/pathogens13060440

APA Style

Fernandez-Baca, M. V., Castellanos-Gonzalez, A., Ore, R. A., Alccacontor-Munoz, J. L., Hoban, C., Castro, C. A., Tanabe, M. B., Morales, M. L., Ortiz, P., White, A. C., Jr., Cabada, M. M., & on behalf of the Fasciola TMRC in Peru. (2024). A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens. Pathogens, 13(6), 440. https://doi.org/10.3390/pathogens13060440

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