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Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp.
Sören Hansen1, Jenny Schäfer1, Kim Fechner1
1Division of Microbiology and Animal Hygiene, Institute of Veterinary Medicine, Department of Animal Sciences, Faculty of Agricultural Sciences, Georg-August University, Goettingen, Germany.
Background:
The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.
Methodology/Principal Findings:
In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.
Conclusions/Significance:
The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.
Insights
A new rapid recombinase polymerase amplification (RPA) assay detects Mycobacterium avium subsp. paratuberculosis (MAP) in 15 minutes. This assay offers high specificity and sensitivity for MAP detection in ruminants, improving disease control.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Infectious Disease Control
Background:
- Accurate detection of Mycobacterium avium subsp. paratuberculosis (MAP) is vital for controlling its spread in ruminants and preventing human transmission.
- Traditional MAP detection methods like cultivation are time-consuming, while existing PCR assays require complex equipment and procedures.
- There is a need for rapid, accessible diagnostic tools for MAP infections.
Purpose of the Study:
- To develop and evaluate a rapid diagnostic assay for Mycobacterium avium subsp. paratuberculosis (MAP) detection.
- To assess the performance of a recombinase polymerase amplification (RPA) assay targeting the IS900 gene for MAP identification.
Main Methods:
- Development of a recombinase polymerase amplification (RPA) assay targeting the IS900 gene specific to MAP.
- Determination of the assay's detection limit using plasmid standards and probit analysis.
- Evaluation of clinical performance using blood, sperm, fecal, and tissue samples from MAP-infected and non-infected ruminants, compared against real-time PCR.
Main Results:
- The MAP RPA assay achieved a detection limit of 16 DNA molecules in 15 minutes.
- No cross-reactivity was observed with other mycobacterial or bacterial strains.
- The assay demonstrated 100% specificity and 89.5% sensitivity when tested with clinical samples.
Conclusions:
- The developed RPA assay provides a rapid, user-friendly alternative to traditional MAP detection methods.
- Reagents are cold-chain independent, enhancing its utility in various settings.
- Combined with a simple extraction protocol, the RPA assay is suitable for point-of-need MAP detection.
