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Published on: September 14, 2011
A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium
Martina Cechova1,2, Monika Beinhauerova3,4, Vladimir Babak3
1Department of Microbiology and Antimicrobial Resistance, Veterinary Research Institute, Brno, Czech Republic. martina.cechova@vri.cz.
Abstract:
Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.
Insights
A new palladium (Pd)-based quantitative PCR (qPCR) method offers a faster, simpler way to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) cells. This Pd-qPCR assay avoids the complex procedures of propidium monoazide (PMA)-qPCR, improving cell viability assessment.
Area of Science:
- Microbiology
- Molecular Biology
- Veterinary Science
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis in ruminants.
- Current MAP detection relies on slow culture methods or complex propidium monoazide quantitative PCR (PMA-qPCR).
- PMA-qPCR requires darkroom and ice-based procedures, limiting its practical application.
Purpose of the Study:
- To optimize a simpler, faster viability assay for MAP using palladium (Pd) compounds.
- To compare the efficacy of the new Pd-based assay with existing PMA-qPCR methods.
- To establish Pd-qPCR as a viable alternative for detecting live MAP cells.
Main Methods:
- Optimized a quantitative PCR (qPCR) assay using palladium (Pd) compounds (bis(benzonitrile)dichloropalladium(II) or palladium(II)acetate) for sample treatment.
- Exposed MAP cells to 30 µM Pd compounds at 5°C for 90 minutes in ultrapure water.
- Compared quantification cycle (Cq) values between live and dead MAP cells treated with Pd compounds and PMA.
Main Results:
- The optimized Pd-qPCR assay showed significant differences in Cq values between live and dead MAP cells (8.5 and 7.9 for the two Pd compounds, respectively).
- These differences correspond to approximately 2.5 log units, indicating effective differentiation of viable cells.
- Pd-qPCR demonstrated superior performance compared to PMA-qPCR in distinguishing live from dead MAP cells.
Conclusions:
- The developed Pd-qPCR assay is a robust and efficient method for detecting viable MAP cells.
- This assay offers a less laborious alternative to PMA-qPCR and faster results than culture methods.
- Pd-qPCR has potential applications in food, feed, clinical, and environmental sample analysis for viable MAP detection.
