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Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens
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Development of a novel LAMP diagnostic method for visible detection of swine Pasteurella multocida
Dongbo Sun1, Jianfa Wang, Rui Wu
1College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing High-tech Industrial Development Zone, Daqing, 163319, People's Republic of China. dongbosun@yahoo.com.cn
Abstract:
A set of four specific primers for six regions of kmt1 gene from a species specific region was designed for developing the loop-mediated isothermal amplification diagnostic method of swine Pasteurella multocida (Pm-LAMP). After the Pm-LAMP was carried out at 63°C for 1 h, the LAMP products could be visually confirmed using fluorescent dyes as detection reagent under UV-illumination. In sensitivity, the detection limit of the Pm-LAMP was 10 cfu/mL, and was 1 log less than that of the PCR method. In specificity, the Pm-LAMP did not amplify genomic DNA of swine common respiratory pathogens. Furthermore, based on results for clinical swab samples (n = 31) using PCR detection as golden standard, relative sensitivity of the Pm-LAMP was 100%, relative specificity of the Pm-LAMP was 90.9%, and percentage of observation agreement was 93.5% (Kappa = 0.85). The Pm-LAMP method should be a useful diagnostic tool for rapid and visible detection of swine Pasteurella multocida.
Insights
A new loop-mediated isothermal amplification (LAMP) method for swine Pasteurella multocida detection is developed. This rapid, visible diagnostic tool offers high sensitivity and specificity for swine respiratory disease management.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacteriology
Background:
- Swine Pasteurella multocida (Pm) causes significant respiratory disease in pigs.
- Accurate and rapid diagnostic methods are crucial for effective disease control.
- Existing methods like PCR require specialized equipment and trained personnel.
Purpose of the Study:
- To develop a novel, rapid, and visually detectable diagnostic method for swine Pasteurella multocida.
- To establish a loop-mediated isothermal amplification (LAMP) assay for specific detection of Pm.
- To evaluate the sensitivity, specificity, and clinical applicability of the developed Pm-LAMP assay.
Main Methods:
- Design of four specific primers targeting six regions of the kmt1 gene for swine Pasteurella multocida.
- Optimization of the loop-mediated isothermal amplification (LAMP) reaction conditions (63°C for 1 hour).
- Visual detection of LAMP products using fluorescent dyes under UV illumination.
- Validation using PCR as the gold standard on clinical swab samples (n=31).
Main Results:
- The developed Pm-LAMP assay achieved a detection limit of 10 cfu/mL, which is 1 log lower than PCR.
- The assay demonstrated high specificity, showing no amplification with DNA from other common swine respiratory pathogens.
- Clinical sample testing revealed a relative sensitivity of 100%, relative specificity of 90.9%, and 93.5% agreement (Kappa = 0.85) compared to PCR.
Conclusions:
- The developed Pm-LAMP method provides a rapid, sensitive, and visually detectable diagnostic tool for swine Pasteurella multocida.
- This method is suitable for field applications and resource-limited settings due to its simplicity and visual readout.
- The Pm-LAMP assay can aid in timely diagnosis and management of swine respiratory infections caused by Pasteurella multocida.
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