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Related Concept Videos

Pulmonary Tuberculosis IV01:26

Pulmonary Tuberculosis IV

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Tuberculosis, more commonly referred to as TB, is an infectious disease stemming from Mycobacterium tuberculosis. While it primarily impacts the lungs, TB can also affect other body areas. Given its severity and global impact, timely and accurate diagnosis is crucial for controlling its spread and improving patient outcomes.
Several diagnostic approaches are used to detect TB. The conventional method is the Tuberculin Skin Test (TST), also known as the Mantoux test. However, this method has...
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Related Experiment Video

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Identification of Mycobacterium Species by DNA Microarray Chip Method
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Direct PCR on Tissue Samples To Detect Mycobacterium tuberculosis Complex: an Alternative to the Bacteriological

V Lorente-Leal1,2, E Liandris1, M Pacciarini3

  • 1VISAVET Health Surveillance Center, Complutense University of Madrid, Madrid, Spain.

Journal of Clinical Microbiology
|November 26, 2020
PubMed
Summary

A new real-time PCR test targeting the IS6110 transposon shows high accuracy for detecting bovine tuberculosis (bTB) in cattle tissues. This rapid method offers a valuable alternative to traditional culture techniques for bTB eradication programs.

Keywords:
PCRbovine tuberculosisdirect PCRtissue samplesvalidation

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Area of Science:

  • Veterinary Microbiology
  • Molecular Diagnostics
  • Infectious Disease Control

Background:

  • Bovine tuberculosis (bTB) remains a significant concern in the European Union, impacting cattle health and trade.
  • Current diagnostic methods, primarily microbiological culture, suffer from limitations including moderate sensitivity and lengthy incubation periods.
  • There is a critical need for more sensitive and rapid diagnostic tools to support bTB eradication efforts.

Purpose of the Study:

  • To evaluate the analytical and diagnostic performance of a real-time PCR assay targeting the IS6110 transposon for detecting Mycobacterium tuberculosis complex (MTBC) in bovine tissues.
  • To assess the robustness and repeatability of the real-time PCR assay through an interlaboratory trial involving EU National Reference Laboratories.
  • To compare the real-time PCR assay's performance against the gold standard microbiological culture method.

Main Methods:

  • A real-time PCR assay targeting the MTBC-specific IS6110 transposon was developed and validated.
  • Analytical performance was assessed by determining the limit of detection (LOD).
  • Diagnostic performance was evaluated using bovine tissue samples from the Spanish bTB eradication campaign, comparing results to microbiological culture. An interlaboratory trial was conducted for robustness and repeatability assessment.

Main Results:

  • The real-time PCR assay demonstrated a limit of detection of 65 fg/reaction of purified genomic equivalents.
  • Diagnostic sensitivity (Se) was 96.45% and diagnostic specificity (Sp) was 93.66%, with an overall agreement (κ) of 0.88 when compared to culture.
  • Cross-reactivity was observed with Mycobacterium marinum and Mycobacterium avium subsp. hominissuis, the latter containing an IS6110-like sequence.

Conclusions:

  • The real-time PCR assay targeting IS6110 is a highly sensitive and specific diagnostic tool for bovine tuberculosis detection in cattle.
  • Despite minor cross-reactivity issues, the assay represents a valuable and rapid alternative to conventional microbiological culture.
  • The implementation strategy of this molecular method should be tailored to specific national or regional bTB control and eradication programs.