Quantification of Viable Pseudomonas species based on DNA Amplification
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Abstract
PCR techniques have significantly improved the detection and quantification of bacterial pathogens on foods. However, there are still limitations in the use of PCR-based diagnostics; the lack of discrimination between DNA derived from viable and dead microorganisms is a major obstacle of the PCR method. Ethidium bromide monoazide is a DNA binding dye that differentiate viable and dead cells. However, there are reports that EMA at higher concentration can equally reduce or inhibit DNA amplification of viable cells. The aim of this study was to determine the concentration of EMA that will inhibit DNA amplification from dead Pseudomonas species but allow amplification of only viable Pseudomonas species by using EMA in conjunction with the conventional PCR.
In this study, we have developed the EMA-PCR assay for the detection and quantification of viable Pseudomonas species. This study shows that 1.0 µg/ml EMA inhibit DNA amplification derived from dead Pseudomonas species cell with no significant (P<0.05) inhibition from viable cells. The EMA-PCR assay can detect as few as 1CFU/PCR viable cells target DNA in a mixed ratio of viable-dead cells suspensions. This EMA-PCR assay offers the advantage of obtaining results in 2 to 4 h compared to 48 h from conventional plate counts and is sensitive and reliable for comprehensive detection and quantification of Pseudomonas species that could be used in private sectors and food industries for monitoring food products to identify potential risk.