RT Journal Article
T1 Validation of a real-time PCR for the detection of mycobacterium tuberculosis complex members in Bovine tissue samples
A1 Lorente Leal, Víctor
A1 Liandris, Emmanouil
A1 Castellanos, Elena
A1 Bezos Garrido, Javier
A1 Domínguez Rodríguez, Lucas José
A1 Juan Ferré, Lucía De
A1 Romero Martínez, Beatriz
AB Although the post-mortem diagnosis of bovine tuberculosis is mainly achieved through microbiological culture, the development of other techniques to detect Mycobacterium tuberculosis complex (MTBC) members directly from tissue samples has been pursued. The present study describes the development, optimization and validation of a Real-Time PCR based on the mpb70 gene to detect MTBC members in clinical tissue samples from cattle. Specific primers and a hybridization probe were used to amplify MTBC-specific sequences in order to avoid cross-reaction with non-MTBC species. An Internal Amplification Control (IAC) was included in order to assess the presence of PCR inhibitors in the samples. The PCR was optimized to achieve maximum efficiency, and the limit of detection, limit of quantification and dynamic range of the reaction were determined. The specificity of the reaction was tested against 34 mycobacterial and non-mycobacterial species. The diagnostic sensitivity, specificity and positive and negative predictive values (PPV and NPV) of the method were assessed on 200 bovine tissue samples in relation to bacteriological culture. The dynamic range of the reaction spanned from 5 ng/reaction (106 genome equivalents) to 50 fg/reaction (10 genome equivalents). The efficiency of the reaction was 102.6% and the achieved R2 was 0.999. The limit of detection with 95% confidence was 10 genome equivalents/reaction. No cross-reactions with non-MTBC species were observed. The diagnostic sensitivity and specificity values of the mpb70 specific Real-Time PCR respect to culture were 94.59% (95% CI: 86.73–98.51%) and 96.03% (95% CI: 90.98–98.70%), respectively, with a PPV of 93.33% (95% CI: 85.55–97.07%) and a NPV of 96.80% (95% CI: 92.10–98.74%). The concordance of the Real-Time PCR based on mpb70 is comparable to that of culture (K = 0.904) showing a great potential for the detection of members of the MTBC in animal tissues.
PB Frontiers Media
YR 2019
FD 2019
LK https://hdl.handle.net/20.500.14352/133259
UL https://hdl.handle.net/20.500.14352/133259
LA eng
NO Lorente-Leal, V., Liandris, E., Castellanos, E., Bezos, J., Domínguez, L., de Juan, L., & Romero, B. (2019). Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples. Frontiers in veterinary science, 6, 61. https://doi.org/10.3389/fvets.2019.00061
NO VL-L and EL performed all experiments in this study and the in silico specificity analysis. EC participated in the design of the mpb70 specific oligonucleotides used in this study. BR, EL, and LdJ designed the study. LD and LdJ are responsible for the obtaining of samples. VL-L wrote the manuscript with the invaluable insights of EC, JB, EL, LD, BR, and LdJ. BR directed and supervised the complete study.Becas/contratos:Víctor Lorente Leal: predoctoral grant from the Complutense University of Madrid and Banco Santander 2017–2018.
NO Banco Santander
NO Universidad Complutense de Madrid
DS Docta Complutense
RD 18 mar 2026