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Publications

Journal Article Epublish 3 citations

PubMed: 37623978 DOI: 10.3390/pathogens12081018

The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Species.

Pathogens 12(8)

Authors: David Vasco-Julio, MarĂ­a Huilca-Ibarra, Yanua Ledesma, Gustavo Echeverria, Salome Guerrero-Freire, Tomasz Jagielski, Carlos Bastidas-Caldes, Jacobus H de Waard

Affiliations

  • One Health Research Group, Universidad de Las AmĂ©ricas, Quito 170530, Ecuador.
  • Posgrado en Ciencias BiolĂ³gicas, Universidad Nacional AutĂ³noma de MĂ©xico, Unidad de Posgrado, Edificio D, Circuito de Posgrados, Ciudad Universitaria, CoyoacĂ¡n C.P. 04510, Mexico.
  • Centro de InvestigaciĂ³n Sobre Enfermedades Infecciosas, Instituto Nacional de Salud PĂºblica, Cuernavaca C.P. 62050, Mexico.
  • Instituto de InvestigaciĂ³n en Zoonosis-CIZ, Universidad Central del Ecuador, Quito 170518, Ecuador.
  • DivisiĂ³n InvestigaciĂ³n y Desarrollo, BioGENA, Quito 170509, Ecuador.
  • Programa de Doctorado, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires C1063ACV, Argentina.
  • Group of Emerging and Neglected Diseases, Ecoepidemiology and Biodiversity, Health Sciences Faculty, Universidad Internacional SEK, Quito 170521, Ecuador.
  • Department of Medical Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland.
  • INABIO-Instituto Nacional de Biodiversidad, Parque La Carolina, Quito 170135, Ecuador.

Abstract

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic species, including the three species associated with infection in dairy cattle (, , and ) and the two species associated with human infections ( and ). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published cytochrome B sequences and was evaluated using reference strains from each of the five species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three species, providing a rapid test that delivers results within three hours.

MeSH

Infections, Multiplex Polymerase Chain Reaction