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Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida
Abstract:
OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay). SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations. PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HS-LAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains. RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HS-LAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.
Insights
Developed rapid loop-mediated isothermal amplification (LAMP) assays detect Pasteurella multocida DNA. The hemorrhagic septicemia (HS)-LAMP assay showed high sensitivity and specificity for identifying HS-associated P multocida strains.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacteriology
Background:
- Pasteurella multocida is a significant bacterial pathogen causing various diseases in animals.
- Hemorrhagic septicemia (HS) is a severe, often fatal, disease in cattle and buffalo caused by specific strains of P. multocida.
- Accurate and rapid diagnostic methods are crucial for controlling HS outbreaks.
Purpose of the Study:
- To develop and evaluate two rapid loop-mediated isothermal amplification (LAMP) assays.
- To detect Pasteurella multocida DNA (Pm-LAMP assay).
- To detect P. multocida DNA specifically from strains associated with hemorrhagic septicemia (HS-LAMP assay).
Main Methods:
- Optimization of reaction conditions for both Pm-LAMP and HS-LAMP assays.
- Detection of bacterial DNA using the developed LAMP assays.
- Validation of LAMP assay results against conventional PCR methods for P. multocida and HS-associated serotypes.
Main Results:
- The HS-LAMP assay demonstrated high specificity (93-100%) and sensitivity (97-100%) at low template DNA concentrations (5 pg) within 27-28 minutes.
- The Pm-LAMP assay achieved optimal specificity (93-97%) and sensitivity (91-98%) at 10 pg DNA within 33-34 minutes.
- Both assays showed promising results when compared to conventional PCR.
Conclusions:
- The developed HS-LAMP assay exhibits high sensitivity and specificity for detecting HS-associated P. multocida.
- These findings suggest the potential utility of the HS-LAMP assay for rapid diagnosis.
- Further research is required to assess the assay's accuracy on clinical samples from endemic regions.
