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Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
Determination of viable Salmonellae from potable and source water through PMA assisted qPCR
Gulshan Singh1, Poornima Vajpayee, Saurabh Bhatti
1Environmental Microbiology, Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research, Post Box 80, Mahatma Gandhi Marg, Lucknow-226001, Uttar Pradesh, India.
Abstract:
Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management.
Insights
This study introduces a new method using propidium monoazide (PMA) with qPCR to accurately detect viable Salmonella in water. This improves microbial risk management in water distribution systems.
Area of Science:
- Environmental microbiology
- Molecular biology
- Public health
Background:
- Salmonella infections pose a significant economic burden in resource-limited endemic regions.
- Conventional qPCR for Salmonella detection cannot differentiate between live and dead bacterial cells.
Purpose of the Study:
- To develop and validate a method for quantifying viable Salmonella in water sources.
- To assess the presence of viable Salmonella in urban water supplies in North India.
Main Methods:
- Coupling propidium monoazide (PMA) with ttr gene-specific quantitative polymerase chain reaction (qPCR).
- PMA selectively penetrates nonviable cells, cross-linking their DNA for exclusion from analysis.
- Analysis of viable Salmonella in source water and urban potable water samples.
Main Results:
- Viable Salmonella were detected in source water (2.1×10(4)-2.6×10(6) CFU/100mL) and potable water (2-7160 CFU/100mL).
- Potable water at the treatment facility showed DNA from dead cells but no viable Salmonella.
- PMA-assisted qPCR successfully detected low levels of live Salmonella in both water types.
Conclusions:
- PMA-assisted qPCR provides specific detection of viable Salmonella in water.
- This method is valuable for surveillance of potable water distribution networks.
- The strategy aids in mapping contamination points for enhanced microbial risk management.
