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A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays
Hannah B Pooley1, Kumudika de Silva1, Auriol C Purdie1
1Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
Applied and Environmental Microbiology
|July 3, 2016
Summary
A new quantitative PCR method rapidly quantifies viable Mycobacterium avium subsp. paratuberculosis in 2 weeks, unlike traditional culture methods taking 4 months. This accelerates bacterial viability testing for research and development.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Bacterial viability determination is crucial for in vitro cellular infection assays.
- Traditional culture methods for Mycobacterium avium subsp. paratuberculosis are slow (up to 4 months), hindering research.
- Fastidious and slow-growing bacteria pose significant challenges in viability assessment.
Purpose of the Study:
- To identify a rapid assay for quantifying viable Mycobacterium avium subsp. paratuberculosis in cellular samples.
- To overcome the limitations of slow culture-based methods in bacterial viability testing.
- To enable large-scale screening for vaccine and antimicrobial development.
Main Methods:
- Assessed three commercial bacterial viability assays.
- Developed a modified liquid culture method combined with quantitative PCR (qPCR).
- Evaluated assay performance based on differentiation of live/dead bacteria and accuracy at low concentrations.
Main Results:
- The qPCR-coupled liquid culture method reliably detected and quantified M. avium subsp. paratuberculosis growth within 2 weeks.
- A strong linear association was observed between 2-week growth rate and initial bacterial concentration.
- Commercially available viability assays were unsuitable for samples from in vitro cellular infection assays.
Conclusions:
- The developed qPCR and liquid culture method provides rapid and reliable quantification of viable M. avium subsp. paratuberculosis.
- This method significantly reduces assay time from months to weeks, facilitating high-throughput screening.
- Accelerated viability testing is essential for advancing vaccine and antimicrobial development for M. avium subsp. paratuberculosis infections.
