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Rapid and Specific Detection of B. melitensis Targeting BMEI1661 Gene Using Loop-mediated Isothermal Amplification
Marcia Ashmi1, Bablu Kumar2, Sanjana1
1Division of Biological Products, ICAR-Indian Veterinary Research Institute, Bareilly, UP, India.
Abstract:
B. melitensis is the most pathogenic zoonotic species of Brucella transmitted to animals through fetal secretions, placenta, and vaginal discharges of infected animals and humans by ingesting unpasteurized milk, dairy products, and raw meat. Early detection of B. melitensis is essential for timely intervention and control of the disease. The gold standard diagnostic methods, such as culture, are time-consuming and may take several weeks aiding to the disease spread. Loop-mediated isothermal amplification assay (LAMP) is widely used to detect infectious pathogens. LAMP can be utilized as a rapid point-of-care test, but has lower specificity which can be enhanced by combining this test with lateral flow immunoassay. No point-of-care test is available for detecting Brucella melitensis in clinical samples. Herein, we developed a LAMP coupled with lateral flow immunoassay (LFIA) for the specific detection of B. melitensis. The sensitivity of LAMP-LFIA was found to be 12.1 fg of genomic DNA isolated from the organism, which is 100-fold more sensitive to conventional PCR and equally sensitive to Real-time (RT-PCR). Moreover, the assay demonstrated high specificity when tested against other Brucella and non-Brucella species. The infective dose of B. melitensis is relatively low for humans, which may remain undetected by conventional PCR, but will be detected using the new technique.
Insights
A new diagnostic test combining loop-mediated isothermal amplification (LAMP) and lateral flow immunoassay (LFIA) allows for rapid and specific detection of Brucella melitensis. This point-of-care test is highly sensitive, improving early disease detection and control.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Brucella melitensis is a highly pathogenic zoonotic bacterium causing significant animal and human health concerns.
- Traditional diagnostic methods like culture are slow, hindering timely disease intervention and control.
- Existing rapid tests lack the specificity required for reliable clinical detection of B. melitensis.
Purpose of the Study:
- To develop and validate a novel point-of-care diagnostic assay for the specific detection of Brucella melitensis.
- To enhance the sensitivity and specificity of pathogen detection compared to existing methods.
- To provide a tool for early and accurate diagnosis of B. melitensis infections in clinical settings.
Main Methods:
- Development of a loop-mediated isothermal amplification (LAMP) assay targeting B. melitensis.
- Integration of the LAMP assay with a lateral flow immunoassay (LFIA) for enhanced specificity and visual detection.
- Validation of the LAMP-LFIA assay using genomic DNA and comparison with conventional PCR and real-time PCR.
Main Results:
- The developed LAMP-LFIA assay demonstrated high sensitivity, detecting as little as 12.1 fg of B. melitensis genomic DNA.
- The assay exhibited superior sensitivity compared to conventional PCR (100-fold) and comparable sensitivity to real-time PCR.
- High specificity was confirmed against a panel of other Brucella and non-Brucella species, ensuring accurate identification.
Conclusions:
- The novel LAMP-LFIA assay offers a rapid, sensitive, and specific point-of-care method for detecting Brucella melitensis.
- This diagnostic tool can detect low infective doses of B. melitensis, which may be missed by conventional PCR.
- The assay facilitates timely intervention and improved control strategies for brucellosis in both animal and human health.
