Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples
Brucellosis is a major infectious disease of cattle. It is also an international trade barrier for the import and export of dairy and beef products. In Mexico, bovine brucellosis is diagnosed using the card, rivanol, and complement fixation serological tests. Molecular methods such as polymerase chain reaction (PCR) are rapid, specific diagnostic tools for brucellosis. This research developed a duplex PCR assay for the diagnosis of brucellosis in cattle blood samples, using the omp2 and BSCP31 genes. Fifty three (53) blood samples with rivanol titers of 1:400, and 60 serologically-negative samples were used. The optimum concentrations of both primers and magnesium chloride for specific fragment amplifications were 100 nM and 0.5 mM, respectively. The analytical sensitivity of duplex PCR was 100 fg/μ,,,,l DNA, while the optimum amplification concentration was 1 ng/μ,,,,l DNA. Analytical specificity was 100%. Diagnostic sensitivity and specificity were 96.3% and 100%, respectively. The results of this study provide evidence for the routine use of duplex PCR in the diagnosis of bovine brucellosis directly on blood samples, as a highly safe, sensitive, specific method.
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Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias
2012
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rev-remexcp-article18142020-06-18T19:43:54Z Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples Diagnóstico rápido y efectivo de brucelosis bovina en sangre, mediante una reacción en cadena de la polimerasa doble Aguirre Arzola, Víctor Eustorgio Alvarado González, Mónica Ibave González, José Luís Leal Hernández, Marisela Díaz Aparicio, Efrén Nevárez Moorillón, Guadalupe Virginia Solís Martínez, Francisco Javier Arévalo Gallegos, Sigifredo Estela River, Blanca Cattle, Brucellosis, Duplex PCR, Blood Bovinos, Brucelosis, PCR doble, Sangre Brucellosis is a major infectious disease of cattle. It is also an international trade barrier for the import and export of dairy and beef products. In Mexico, bovine brucellosis is diagnosed using the card, rivanol, and complement fixation serological tests. Molecular methods such as polymerase chain reaction (PCR) are rapid, specific diagnostic tools for brucellosis. This research developed a duplex PCR assay for the diagnosis of brucellosis in cattle blood samples, using the omp2 and BSCP31 genes. Fifty three (53) blood samples with rivanol titers of 1:400, and 60 serologically-negative samples were used. The optimum concentrations of both primers and magnesium chloride for specific fragment amplifications were 100 nM and 0.5 mM, respectively. The analytical sensitivity of duplex PCR was 100 fg/μ,,,,l DNA, while the optimum amplification concentration was 1 ng/μ,,,,l DNA. Analytical specificity was 100%. Diagnostic sensitivity and specificity were 96.3% and 100%, respectively. The results of this study provide evidence for the routine use of duplex PCR in the diagnosis of bovine brucellosis directly on blood samples, as a highly safe, sensitive, specific method. La brucelosis es una de las enfermedades infecciosas más importantes del ganado y representa una barrera para la importación y exportación de productos lácteos y cárnicos. En México, el diagnóstico se realiza mediante las pruebas serológicas de tarjeta, rivanol y fijación del complemento. Los métodos moleculares, como la reacción en cadena de la polimerasa (PCR), son herramientas rápidas y específicas para el diagnóstico de la enfermedad. En el presente trabajo se desarrolló el diagnóstico de brucelosis por PCR doble, a partir de muestras de sangre, utilizando los genes omp2 y BSCP31. Para este estudio se utilizaron 53 muestras de sangre con títulos de rivanol de 1:400 y 60 muestras con resultados negativos a las pruebas serológicas. Las concentraciones óptimas de iniciadores y cloruro de magnesio para lograr la amplificación específica de los dos fragmentos, fueron de 100 nM y 0.5 mM respectivamente. La sensibilidad analítica alcanzada para la PCR doble fue de 100 fg/μ,,,,l de ADN, mientras que la concentración óptima de amplificación fue de 1 ng/μ,,,,l de ADN. La especificidad analítica obtenida fue del 100 %, mientras que la sensibilidad y la especificidad diagnóstica fueron del 96.3 % y 100 % respectivamente. Los resultados de este estudio aportan evidencia para el uso rutinario de la PCR doble para el diagnóstico de la brucelosis bovina directamente de muestras de sangre, ya que es un método altamente seguro, sensible y específico. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias 2012-01-01 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/1814 Revista Mexicana de Ciencias Pecuarias; Vol. 46, Núm. 2 (2008); 147 a 158 Revista Mexicana de Ciencias Pecuarias; Vol. 46, Núm. 2 (2008); 147 a 158 2448-6698 2007-1124 spa https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/1814/1808 http://creativecommons.org/licenses/by-nc-sa/4.0 |
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Aguirre Arzola, Víctor Eustorgio Alvarado González, Mónica Ibave González, José Luís Leal Hernández, Marisela Díaz Aparicio, Efrén Nevárez Moorillón, Guadalupe Virginia Solís Martínez, Francisco Javier Arévalo Gallegos, Sigifredo Estela River, Blanca |
| spellingShingle |
Aguirre Arzola, Víctor Eustorgio Alvarado González, Mónica Ibave González, José Luís Leal Hernández, Marisela Díaz Aparicio, Efrén Nevárez Moorillón, Guadalupe Virginia Solís Martínez, Francisco Javier Arévalo Gallegos, Sigifredo Estela River, Blanca Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| author_facet |
Aguirre Arzola, Víctor Eustorgio Alvarado González, Mónica Ibave González, José Luís Leal Hernández, Marisela Díaz Aparicio, Efrén Nevárez Moorillón, Guadalupe Virginia Solís Martínez, Francisco Javier Arévalo Gallegos, Sigifredo Estela River, Blanca |
| author_sort |
Aguirre Arzola, Víctor Eustorgio |
| title |
Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| title_short |
Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| title_full |
Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| title_fullStr |
Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| title_full_unstemmed |
Duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| title_sort |
duplex polymerase chain reaction as a rapid, effective diagnostic test for bovine brucellosis using blood samples |
| description |
Brucellosis is a major infectious disease of cattle. It is also an international trade barrier for the import and export of dairy and beef products. In Mexico, bovine brucellosis is diagnosed using the card, rivanol, and complement fixation serological tests. Molecular methods such as polymerase chain reaction (PCR) are rapid, specific diagnostic tools for brucellosis. This research developed a duplex PCR assay for the diagnosis of brucellosis in cattle blood samples, using the omp2 and BSCP31 genes. Fifty three (53) blood samples with rivanol titers of 1:400, and 60 serologically-negative samples were used. The optimum concentrations of both primers and magnesium chloride for specific fragment amplifications were 100 nM and 0.5 mM, respectively. The analytical sensitivity of duplex PCR was 100 fg/μ,,,,l DNA, while the optimum amplification concentration was 1 ng/μ,,,,l DNA. Analytical specificity was 100%. Diagnostic sensitivity and specificity were 96.3% and 100%, respectively. The results of this study provide evidence for the routine use of duplex PCR in the diagnosis of bovine brucellosis directly on blood samples, as a highly safe, sensitive, specific method. |
| publisher |
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias |
| publishDate |
2012 |
| url |
https://cienciaspecuarias.inifap.gob.mx/index.php/Pecuarias/article/view/1814 |
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