Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina
Bovine babesiosis is a tick-borne disease caused by protozoan parasites of the genus Babesia. Babesia bigemina is one of the most prevalent and economically important parasite species that infects cattle because of its impact on the meat and milk production industry. Effective disease control strate...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2022.1056355/full |
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| author | Andrea P. Lizarazo-Zuluaga Andrea P. Lizarazo-Zuluaga Bertha I. Carvajal-Gamez Bertha I. Carvajal-Gamez Silvina Wilkowsky Silvio Cravero Marcos Trangoni Juan Mosqueda Juan Mosqueda |
| author_facet | Andrea P. Lizarazo-Zuluaga Andrea P. Lizarazo-Zuluaga Bertha I. Carvajal-Gamez Bertha I. Carvajal-Gamez Silvina Wilkowsky Silvio Cravero Marcos Trangoni Juan Mosqueda Juan Mosqueda |
| author_sort | Andrea P. Lizarazo-Zuluaga |
| collection | DOAJ |
| description | Bovine babesiosis is a tick-borne disease caused by protozoan parasites of the genus Babesia. Babesia bigemina is one of the most prevalent and economically important parasite species that infects cattle because of its impact on the meat and milk production industry. Effective disease control strategies should include detection of reservoir animals and early and specific pathogen detection using rapid, economical, sensitive, and specific detection techniques. The loop-mediated isothermal amplification technique (LAMP) is a one-step molecular reaction that amplifies DNA sequences with high sensitivity and specificity under isothermal conditions and requires no special equipment. The results can be observed by the naked eye as color changes. The aim of this work was to develop and standardize the LAMP technique for B. bigemina detection and its visualization using hydroxynaphtol blue. For this situation, primers were designed from the conserved sequences of the B. bigemina ama-1 gene. The results showed that at 63 °C in 1 h and under standardized conditions, this technique could amplify B. bigemina DNA as indicated by the characteristic colorimetric change. Sensitivity evaluation indicated that DNA was amplified at a 0.00000001% parasitemia, and it was demonstrated that this technique specifically amplified the DNA of B. bigemina. Additionally, this technique could amplify DNA from 10 strains of B. bigemina from three different countries. It is concluded that the LAMP technique as modified in our case could specifically amplify B. bigemina DNA and shows high sensitivity, does not cross-react with related organisms, and the product is observed by 60 min of reaction time based on color changes. This report is the first LAMP report that uses sequences that are conserved between strains of the ama-1 gene, demonstrates the results by color changes using hydroxynaphtol blue. We propose LAMP as a rapid and economical alternative method for the molecular detection of B. bigemina. |
| first_indexed | 2024-04-13T21:52:10Z |
| format | Article |
| id | doaj.art-c49666f8e3494accb984d2166f3b9693 |
| institution | Directory Open Access Journal |
| issn | 2297-1769 |
| language | English |
| last_indexed | 2024-04-13T21:52:10Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj.art-c49666f8e3494accb984d2166f3b96932022-12-22T02:28:23ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692022-11-01910.3389/fvets.2022.10563551056355Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigeminaAndrea P. Lizarazo-Zuluaga0Andrea P. Lizarazo-Zuluaga1Bertha I. Carvajal-Gamez2Bertha I. Carvajal-Gamez3Silvina Wilkowsky4Silvio Cravero5Marcos Trangoni6Juan Mosqueda7Juan Mosqueda8Immunology and Vaccines Laboratory, C. A. Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Queretaro, MexicoMaestria en Salud y Producción Animal Sustentable. Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, MexicoImmunology and Vaccines Laboratory, C. A. Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Queretaro, MexicoC.A. Salud Animal y Microbiologia Ambiental. Facultad de Ciencias Naturales, Universidad Autonoma de Queretaro, Queretaro, MexicoInstituto de Agrobiotecnología y Biología Molecular (IABIMO). Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Hurlingham, Buenos Aires, ArgentinaInstituto de Agrobiotecnología y Biología Molecular (IABIMO). Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Hurlingham, Buenos Aires, ArgentinaInstituto de Agrobiotecnología y Biología Molecular (IABIMO). Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Hurlingham, Buenos Aires, ArgentinaImmunology and Vaccines Laboratory, C. A. Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Queretaro, MexicoC.A. Salud Animal y Microbiologia Ambiental. Facultad de Ciencias Naturales, Universidad Autonoma de Queretaro, Queretaro, MexicoBovine babesiosis is a tick-borne disease caused by protozoan parasites of the genus Babesia. Babesia bigemina is one of the most prevalent and economically important parasite species that infects cattle because of its impact on the meat and milk production industry. Effective disease control strategies should include detection of reservoir animals and early and specific pathogen detection using rapid, economical, sensitive, and specific detection techniques. The loop-mediated isothermal amplification technique (LAMP) is a one-step molecular reaction that amplifies DNA sequences with high sensitivity and specificity under isothermal conditions and requires no special equipment. The results can be observed by the naked eye as color changes. The aim of this work was to develop and standardize the LAMP technique for B. bigemina detection and its visualization using hydroxynaphtol blue. For this situation, primers were designed from the conserved sequences of the B. bigemina ama-1 gene. The results showed that at 63 °C in 1 h and under standardized conditions, this technique could amplify B. bigemina DNA as indicated by the characteristic colorimetric change. Sensitivity evaluation indicated that DNA was amplified at a 0.00000001% parasitemia, and it was demonstrated that this technique specifically amplified the DNA of B. bigemina. Additionally, this technique could amplify DNA from 10 strains of B. bigemina from three different countries. It is concluded that the LAMP technique as modified in our case could specifically amplify B. bigemina DNA and shows high sensitivity, does not cross-react with related organisms, and the product is observed by 60 min of reaction time based on color changes. This report is the first LAMP report that uses sequences that are conserved between strains of the ama-1 gene, demonstrates the results by color changes using hydroxynaphtol blue. We propose LAMP as a rapid and economical alternative method for the molecular detection of B. bigemina.https://www.frontiersin.org/articles/10.3389/fvets.2022.1056355/fullBabesia bigeminamolecular detectionLAMPhydroxynaphtol bluediagnostics |
| spellingShingle | Andrea P. Lizarazo-Zuluaga Andrea P. Lizarazo-Zuluaga Bertha I. Carvajal-Gamez Bertha I. Carvajal-Gamez Silvina Wilkowsky Silvio Cravero Marcos Trangoni Juan Mosqueda Juan Mosqueda Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina Frontiers in Veterinary Science Babesia bigemina molecular detection LAMP hydroxynaphtol blue diagnostics |
| title | Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina |
| title_full | Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina |
| title_fullStr | Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina |
| title_full_unstemmed | Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina |
| title_short | Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina |
| title_sort | development and standardization of a loop mediated isothermal amplification lamp test for the detection of babesia bigemina |
| topic | Babesia bigemina molecular detection LAMP hydroxynaphtol blue diagnostics |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2022.1056355/full |
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