Shaken, not stirred: magnetic bead DNA extraction as a rapid and effective method for the scaling up of bovine tuberculosis diagnosis.

Autor: Lorente-Leal V; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain. vicloren@ucm.es., Gomez-Buendia A; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain.; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain., Gutiérrez-Tobaruela A; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain., de Juan L; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain.; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain., Bezos J; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain.; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain., Romero B; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain.; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.
Jazyk: angličtina
Zdroj: BMC veterinary research [BMC Vet Res] 2024 Dec 19; Vol. 20 (1), pp. 568. Date of Electronic Publication: 2024 Dec 19.
DOI: 10.1186/s12917-024-04427-8
Abstrakt: Background: The growing use of real-time PCR (qPCR) as a diagnostic method for bovine TB (bTB) requires rapid and effective DNA extraction methods, which are crucial for its success. Automated DNA extraction methods based on magnetic beads are a promising alternative to conventional silica column-based protocols (COL protocol) due to their high throughput capacity and reduced hands-on time. This study aimed to assess the performance of the MagMax CORE Nucleic Acid Purification kit and the KingFisher Flex instrument (KF protocol) as an alternative for scaling up the use of qPCR in bTB diagnosis.
Methodology: Performance was evaluated with two different real-time PCR (qPCR) protocols, based on the IS6110 element and the QuantiFast and VetMAX™ (QF and VM protocols) kits, on 145 frozen tissue homogenates confirmed as either bTB-positive or negative through a composite reference standard based on microbiological culture, column-based extraction, and qPCR, as well as on negative tissue samples spiked with 10 6 to 10 3 CFU/ml of M. bovis BCG.
Results: The performance of both qPCR protocols was very high on samples extracted using the KF protocol, with positive percent agreement (PPA) values of 89.04% [95% Confidence Interval (CI): 79.54-95.15%] and 93.15% [95% CI: 84.74-97.74%] for the QF and VM protocols, respectively, and negative percent agreement (NPA) values of 100% [95% CI: 95.01-100.00%]. A higher variability was identified in samples analysed with the same qPCR protocol but different extraction methods. Higher Ct values were identified for samples extracted using the KF protocol in both routine and spiked samples, likely due to using the same amount of starting material for both extraction methods, which was lower than recommended by the manufacturer for the KF protocol.
Discussion: The results of this study indicate that the MagMAX CORE Nucleic Acid Purification kit coupled with a KingFisher Flex instrument is a valuable alternative for the extraction of MTBC DNA from bovine tissues. However, the increased variability and Ct values suggest that a larger amount of starting material is recommended for this methodology, warranting further studies.
Competing Interests: Declarations. Ethics approval and consent to participate: All samples were obtained as part of the routine diagnosis workflow of the Spanish bTB eradication programme and, therefore, no animal was sacrificed for the specific purpose of this research study. No ethics approval is therefore required. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
(© 2024. The Author(s).)
Databáze: MEDLINE
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