Related Experiment Video
Updated: Aug 9, 2026
08:10
Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test
Published on: April 9, 2012
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.
Related Concept Videos
02:57Real Time RT-PCR
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The real-time quantification of the number of amplified products is...
01:26Pulmonary Tuberculosis IV
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...
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...