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A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification
Yuji Kouzaki1, Takuya Maeda1, Hiroaki Sasaki1
1Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
Plos One
|July 25, 2015
Summary
A new loop-mediated amplification (LAMP) assay rapidly identifies Bacillus Calmette-Guérin (BCG) by detecting specific DNA deletions. This method is crucial for distinguishing BCG from Mycobacterium tuberculosis, especially in immunocompromised patients.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine against Mycobacterium tuberculosis and used in bladder cancer treatment.
- Distinguishing BCG from other Mycobacterium tuberculosis complexes is critical due to potential severe infections in immunocompromised individuals.
Purpose of the Study:
- To develop a simple, rapid, and specific identification procedure for M. bovis BCG strains.
- To detect deletions unique to M. bovis BCG using loop-mediated amplification (LAMP).
Main Methods:
- Developed a loop-mediated amplification (LAMP) assay targeting a specific deletion region in M. bovis BCG.
- Utilized real-time turbidity and visual color change for amplification detection.
- Employed the Procedure for Ultra Rapid Extraction (PURE) kit for DNA isolation.
- Validated specificity against 49 strains of 44 pathogens, including the M. tuberculosis complex.
Main Results:
- The LAMP assay successfully detected M. bovis BCG-specific deletions.
- Detection limits were 1.0 pg genomic DNA (turbidimeter) and 10 pg (visual inspection).
- Sensitivity reached 1 × 10(3) cells/reaction with the PURE kit in various sample types (serum, urine, CSF, BAL fluid).
- Assay demonstrated high specificity against M. tuberculosis complex and other pathogens.
Conclusions:
- The developed LAMP assay combined with PURE kit offers a rapid, sensitive, and specific method for M. bovis BCG identification.
- This technique is suitable for point-of-care testing, even in resource-limited settings.
- The assay's convenience, speed, and cost-effectiveness make it valuable for clinical settings, particularly for immunocompromised patients.
