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Validation of real-time PCRs for the detection of mycobacteria causing animal tuberculosis

Andrea Ros Carrasco defended this Degree Final Project

May 24th, 2021

Nowadays, animal tuberculosis is a major issue in many countries of the European Union. Although post-mortem detection of this disease is usually performed by microbiological culture, other
methods to detect members of the Mycobacterium tuberculosis complex (MTBC) directly from tissue samples are being developed. One of the most developed and promising technique for this purpose is the polymerase chain reaction (PCR). This work focuses on the optimisation and validation of two Real-Time PCRs for the detection of M. caprae and M. bovis based on the lepA gene and a genomic deletion in the region of difference 4 (RD4), respectively.

The initial objective was to optimise both PCRs for maximum efficiency but only the optimisation for the detection of M. bovis was able to perform. The limit of detection, dynamic range and analytical specificity against 13 species were calculated. Finally, diagnostic specificity and sensitivity were obtained by comparing the PCR results from 52 samples with their culture results. The dynamic range of the reaction was from 5.75 ng/reaction to 5.75 fg/reaction. The reaction efficiency was 96.4% with an R2 of 0.998. The limit of detection with 95% confidence was 1 genomic equivalent/reaction. The diagnostic sensitivity and specificity values against culture were 93.75% (95% CI: 85.4% - 102.1%) and 100%, respectively. The positive predictive value was 100% and the negative predictive value was 90.9% (95% CI: 78.9% - 102.9%). Real-time PCR for RD4 is comparable to culture diagnosis, with a Kappa coefficient of 0.920, which makes this PCR aa great potential protocol for the detection of M. bovis in animal tissues

Link to PhD in Veterinary Medicine

Andrea Ros Carrasco

TITLE: Validación de PCRs tiempo real para la detección de micobacterias causantes de la tuberculosis animal

TYPE: Degree Final Project

AUTHOR: Andrea Ros Carrasco

DIRECTORS: de Juan L. and Romero B.

DATE: May 24th, 2021



Andrea Ros Carrasco. Validación de PCRs tiempo real para la detección de micobacterias causantes de la tuberculosis animal. Universidad Complutense de Madrid. May 24th, 2021. (Degree Final Project)