Endpoint and real-time PCR optimization for detection of Salmonella enterica serovar Gallinarum in poultry from Costa Rica: Detección molecular de Salmonella serotipo Gallinarum en aves de corral

Endpoint and real-time PCR optimization for detection of Salmonella enterica serovar Gallinarum in poultry from Costa Rica

Detección molecular de Salmonella serotipo Gallinarum en aves de corral





molecular detection, DNA, taxonomic placement, Enterobacteriaceae


Introduction: Avian typhoid and pulorosis, diseases caused by Salmonella enterica subsp. enterica serotype Gallinarum biotype Gallinarum and biotype Pullorum, respectively, are responsible for high mortality in poultry, generating significant economic losses for poultry farmers. Objective: To strengthen the detection of the causative agents of Fowl typhoid and pullorum disease by optimizing molecular techniques, such as endpoint PCR and real-time PCR (qPCR). Methods: Bacterial strains control groups, isolates, and tissue from poultry infected with Salmonella Gallinarum were used to standardize the detection by endpoint PCR and qPCR. Results: For the endpoint PCR, a repeatability, specificity, and sensitivity of 100% was obtained, and a Kappa value of 0.98 for reproducibility. With the qPCR, an efficiency of 103% was obtained, while the coefficients of variation for repeatability and reproducibility were lower than 6%. The detection limit of genomic DNA was 6.4 pg/μL, and the number of viable cells was 3x102 CFU/mL for endpoint PCR and 10 copies of DNA per reaction for qPCR. Through sequencing and analysis of the molecular taxonomy of the PCR products, the strains in this study were positioned together with those of serotype S. Gallinarum, corroborating their identity. Conclusion: It was possible to optimize a molecular technique that allows a rapid, reliable, and sensitive detection of Salmonella Gallinarum /Pullorum, which can reduce the waiting time to take action in cases of clinical suspicion and possible outbreaks.


Álvarez, O., Larigauderie, G., Ortega, G. M., Granja, A. C., & Cabria, R. J. (2018). Optimización y Validación de una PCR en Tiempo Real para la Rápida Identificación de Bacillus thuringiensis, Simulador de Bacillus anthracis. Sanidad Militar, 74(2), 84-89.

Barrow, P. A., & Freitas-Neto, O. C. (2011). Pullorum disease and fowl typhoid-new thoughts on old diseases: A review. Avian Pathology, 40(1), 1–13. https://doi.org/10.1080/03079457.2010.542575

Beaubrun, J. J. G., Ewing, L., Dudley, K., Benhamed, F., Wang, H., & Hanes, D. E. (2017). Evaluation of a multiplex PCR method to serotype Salmonella in animal feeds pre-enrichment broth cultures. MethodsX, 4(1), 335-345. https://doi.org/10.1016/j.mex.2017.09.003

Braun, S. D., & Methner, U. (2011). Comparison of DNA isolation methods and detection of Salmonella spp. from animal faeces and dust using invA real- time PCR. Berliner Und Munchener Tierarztliche Wochenschrift, 124(6), 177–185. https://doi.org/10.2376/0005-9366-124-177.

Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular biology and evolution, 17(4), 540-552.

Chen, Y., Ye, W., Zhang, Y., & Xu, Y. (2015). High speed BLASTN: An accelerated MegaBLAST search tool. Nucleic Acids Research, 43(16), 7762–7768. https://doi.org/10.1093/nar/gkv784

Dalai, N., Shekhar, S., Padhy, A., Praveen, P. K., & Sahu, A. R. (2015). Salmonellosis - a potential threat to poultry. Journal of Cell and Tissue Research, 15(3), 5209–5213.

Decreto Ejecutivo No. 34669-MAG (13 de agosto de 2008). Listado de enfermedades animales de declaración obligatoria El presidente de la Republica y el ministro de Agricultura y Ganadería. La Gaceta No.156. http://www.pgrweb.go.cr/scij/Busqueda/Normativa/Normas/nrm_texto_completo.aspx?n Valor1=1&nValor2=63731

Díaz, G., Rosadio, R., Marcelo, G., Chero, A., Jiménez, Ar., Reyna, I., & Maturrano, L. (2017). Evaluación de una Técnica de PCR-Múltiple para la Detección Rápida de Salmonella Typhimurium y Enteritidis en Cuyes (Cavia porcellus) Naturalmente Infectados. Revista de Investigaciones Veterinarias Del Perú, 28(3), 713–722. https://doi.org/http://dx.doi.org/10.15381/rivep.v28i3.13361

Díaz de la Osa, A., Pantoja, M. Q., & Rodríguez, A. H. (2015). Estandarización de la Reacción en Cadena de la Polimerasa con Transcripción Reversa para el diagnóstico molecular de potyvirus que afectan a Capsicum annuum L. en Cuba. Revista CENIC. Ciencias Biológicas, 46(2), 144–155.

Ebentier, D. L., Hanley, K. T., Cao, Y., Badgley, B. D., Boehm, A. B., Ervin, J. S., Goodwin, K. D., Gourmelon, M., Griffith, J.F., Holden, P. A., & Kelthy, C.A. (2013). Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods. Water Research, 47(18), 6839–6848. https://doi.org/10.1016/j.watres.2013.01.060

Foley, S. L., Johnson, T. J., Nayak, R., Ricke, S. C., & Danzeisen, J. (2013). Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars. Microbiology and Molecular Biology Reviews, 77(4), 582–607. https://doi.org/10.1128/mmbr.00015-13

García, R. (2019). Validación de una metodología para la cuantificación de un microorganismo probiótico (Lactobacillus acidophilus La3) en yogur [Tesis de maestría, Universidad de Antioquia, Colombia]. Archivo digital. http://bibliotecadigital.udea.edu.co/handle/10495/11259

Heymans, R., Vila, A., van Heerwaarden, C. A. M., Jansen, C. C. C., Castelijn, G. A. A., van der Voort, M., & Biesta-Peters, E. G. (2018). Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR. Public Library of Science One, 13(10), 1–15. https://doi.org/10.1371/journal.pone.0206316

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: fast model selection for accurate phylogenetic estimates. Nature methods, 14(6), 587-589.

Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution, 30(4), 772-780.

Koetsier, G., & Cantor, E. (2019). A practical guide to analyzing nucleic acid concentration and purity with microvolume spectrophotometers. New England Biolabs Inc. technical note, 8, 7–19. https://www.bioke.com/blobs/downloads/NEB/MVS_Analysis_of_NA_Concentration_and_Purity.pdf

Kralik, P., & Ricchi, M. (2017). A basic guide to real time PCR in microbial diagnostics: Definitions, parameters, and everything. Frontiers in Microbiology, 8(1), 1–9. https://doi.org/10.3389/fmicb.2017.00108

Lavalett, L., Sánchez, M. M., Múñoz, N., Moreno, J., & Cardona-Castro, N. (2009). Desarrollo y validación de una reacción en cadena de la polimerasa múltiple para la identificación de los serogrupos B, C2, D y E de Salmonella entérica. Biomédica, 29(1), 244–252.

Liu, Y., Zhang, D. F., Zhou, X., Xu, L., Zhang, L., & Shi, X. (2017). Comprehensive analysis reveals two distinct evolution patterns of Salmonella flagellin gene clusters. Frontiers in Microbiology, 8(1), 1–12. https://doi.org/10.3389/fmicb.2017.02604

Lorenz, T. C. (2012). Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. Journal of Visualized Experiments, 63(1), 1–15. https://doi.org/10.3791/3998

Minh B.Q., Nguyen, M.A.T., & von Haeseler A. (2013). Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution, 30(5), 1188-1195.

Nguyen, L. T., Schmidt, H. A., Von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular biology and evolution, 32(1), 268-274.

Nurjanah, S., Rahayu, W. P., & Mutaqin, L. Al. (2018). Detection Method for Salmonella Typhimurium and Salmonella Enteritidis using Real-Time Polymerase Chain Reaction. International Journal of Engineering & Technology, 7(1), 302–306.

World Organization for Animal Health (OIE). (2014). Development and optimization of nucleic acid detection assays. Chapter 3.6.3. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris, France: OIE.

World Organization for Animal Health (OIE). (2019). Principles and methods of validation of diagnostic assays for infectious diseases. Chapter 1.1.6. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris, France: OIE.

Pegg, D. E. (2015). Cryopreservation and Freeze-Drying Protocols. Springer Protocols, 1257(1), 477–488. https://doi.org/10.1007/978-1-4939-2193-5

Rambaut, A. (2009). FigTree 2012-2014 (Versión 1.4). http://tree.bio.ed.ac.uk/software/figtree

Secretaría Ejecutiva de Planificación Sectorial Agropecuaria (SEPSA) (2020). Boletín Estadístico Agropecuario: Serie Cronológica 2016-2019. Edición Nº30. http://www.infoagro.go.cr/BEA/BEA30.pdf

Servicio Nacional de Salud Animal (SENASA). (2013). Protocolo Vigilancia de Salmonella en Aves Reproductoras. Programa Nacional Salud Aviar, versión 02. http://www.senasa.go.cr/senasa/sitio/files/231014045057.pdf

Trevethan, R. (2017). Sensitivity, Specificity, and Predictive Values: Foundations, Pliabilities, and Pitfalls in Research and Practice. Frontiers in Public Health, 5(1), 1–7. https://doi.org/10.3389/fpubh.2017.00307

Trifinopoulos, J., Nguyen, L. T., von Haeseler, A., & Minh, B. Q. (2016). W-IQ-TREE: a fast-online phylogenetic tool for maximum likelihood analysis. Nucleic acids research, 44(1), 232-235.

Xiong, D., Song, L., Geng, S., Tao, J., An, S., Pan, Z., & Jiao, X. (2016). One-Step PCR Detection of Salmonella Pullorum/Gallinarum Using a Novel Target: The Flagellar Biosynthesis Gene flhB. Frontiers in Microbiology, 7(1), 1863. https://doi.org/10.3389/fmicb.2016.01863

Xiong, D., Song, L., Pan, Z., & Jiao, X. (2018). Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay. Frontiers in microbiology, 9(1), 1718. https://doi.org/10.3389/fmicb.2018.01718



How to Cite

Leza-Leza, M. T. ., Víquez-Ruiz, E. ., Barquero-Calvo, E. ., Sancho-Blanco, C. ., & Umaña-Castro, R. (2022). Endpoint and real-time PCR optimization for detection of Salmonella enterica serovar Gallinarum in poultry from Costa Rica: Detección molecular de Salmonella serotipo Gallinarum en aves de corral. UNED Research Journal, 14(1), e3831. https://doi.org/10.22458/urj.v14i1.3831