Dataset Information

Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples.

ABSTRACT: 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.

SUBMITTER: Lorente-Leal V

PROVIDER: S-EPMC6409304 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples.

Lorente-Leal Victor V Liandris Emmanouil E Castellanos Elena E Bezos Javier J Domínguez Lucas L de Juan Lucía L Romero Beatriz B

Frontiers in veterinary science 20190304

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 t ...[more]

PMID: 30886855

Similar Datasets

Project description:72 candidate biomarkers evaluated for TB diagnosis using mRNA purified from whole blood of Active TB patients recruited in the UK and India, LTB and two groups of controls from the UK (i) from a low incidence region and (ii) individuals variably domiciled in the UK and India. Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB) and pulmonary (PTB)) and latent TB (LTBI) remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. PBL mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new Tuberculosis diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs)

Dataset Information

Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples.

Publications

Validation of a Real-Time PCR for the Detection of <i>Mycobacterium tuberculosis</i> Complex Members in Bovine Tissue Samples.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets