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Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens
Published on: February 20, 2015
Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella
M Poussard1, S D Pant1,2, J Huang3
1School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, Australia.
Aims:
To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment.
Methods:
Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR.
Results:
A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR.
Conclusion:
The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida.
Clinical Relevance:
The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.
Insights
A new colorimetric loop-mediated isothermal amplification (LAMP) assay effectively detects Pasteurella multocida in poultry. While comparable to PCR, further development is needed for field applications.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Avian Pathology
Background:
- Fowl cholera, caused by Pasteurella multocida, poses a significant threat to poultry health and industry.
- Accurate and rapid diagnostic tools are crucial for controlling disease outbreaks.
- Current diagnostic methods like PCR require specialized laboratory equipment, limiting field applicability.
Purpose of the Study:
- To develop a colorimetric loop-mediated isothermal amplification (LAMP) assay for Pasteurella multocida detection in poultry.
- To compare the diagnostic performance of the developed LAMP assay with conventional PCR.
- To evaluate a simplified DNA extraction method for potential field-based testing.
Main Methods:
- Primer sets targeting the KMT1 gene were designed for both LAMP and PCR assays.
- Analytical sensitivity and specificity were determined using P. multocida isolates and unrelated bacterial species.
- Clinical performance was assessed using cloacal swabs from a commercial turkey flock, comparing a rapid DNA extraction method with a commercial kit.
Main Results:
- The LAMP assay demonstrated 100% analytical specificity and comparable analytical sensitivity (LOD 20 pg) to PCR.
- Clinical testing revealed a lower clinical specificity for LAMP (94.74%) compared to PCR (100%).
- The simplified DNA extraction method was not effective for either LAMP or PCR amplification.
Conclusions:
- The developed colorimetric LAMP assay shows comparable performance to PCR for Pasteurella multocida detection.
- The simplified DNA extraction method requires further optimization for field-based diagnostics.
- LAMP assays, coupled with improved DNA extraction, hold potential for point-of-care testing in field settings for fowl cholera.
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