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Single-tube, nested, reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis
N T Jou1, R B Yoshimori, G R Mason
1Department of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509, USA.
Journal of Clinical Microbiology
|May 1, 1997
Summary
This study introduces a novel reverse transcriptase PCR (RT-PCR) method targeting bacterial mRNA to detect viable Mycobacterium tuberculosis. The assay accurately distinguishes between live and dead bacteria, improving infectious agent detection.
Area of Science:
- Molecular Biology
- Microbiology
- Infectious Diseases
Background:
- Current molecular biology techniques like PCR struggle to differentiate between viable and nonviable infectious agents.
- Bacterial mRNA's short half-life presents a challenge for detection methods.
Purpose of the Study:
- To develop a molecular assay capable of detecting viable Mycobacterium tuberculosis by targeting bacterial mRNA.
- To overcome the limitation of current PCR methods in distinguishing live from dead organisms.
Main Methods:
- Utilized a single-tube, nested, reverse transcriptase PCR (STN RT-PCR) targeting mRNA of the 85B antigen specific to Mycobacterium tuberculosis.
- Developed specific primers for Mycobacterium tuberculosis based on partial gene sequencing.
Main Results:
- The STN RT-PCR detected as few as 12 CFU in clinical sputum specimens and showed sensitivity comparable to culture for smear-negative samples.
- Achieved 100% specificity and successfully differentiated between viable and nonviable Mycobacterium tuberculosis cultures in the presence of isoniazid.
- Demonstrated superiority over DNA PCR in assessing mycobacterial viability.
Conclusions:
- Targeting bacterial mRNA with STN RT-PCR offers a reliable method for detecting viable Mycobacterium tuberculosis.
- This approach significantly enhances the ability to assess the viability of infectious agents, outperforming traditional DNA-based PCR methods.