A Banihashemi1, M I Van Dyke, P M Huck. 1. NSERC Chair in Water Treatment, Department of Civil and Environmental Engineering, University of Waterloo, Waterloo, ON, Canada.
Abstract
AIMS: The effect of amplicon length on the ability of propidium monoazide-PCR (PMA-PCR) to reliably quantify viable cells without interference from dead cells was tested on heat- and ultraviolet (UV)-killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. METHODS AND RESULTS: PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat-treated Salm. enterica (3 log reduction) and Camp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV-irradiated cells did not affect PCR amplification, but long-amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. CONCLUSIONS: The long-amplicon PMA-PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture-based procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane-damaged cells, and also for inactivation mechanisms that cause direct DNA damage.
AIMS: The effect of amplicon length on the ability of propidium monoazide-PCR (PMA-PCR) to reliably quantify viable cells without interference from dead cells was tested on heat- and ultraviolet (UV)-killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. METHODS AND RESULTS: PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat-treated Salm. enterica (3 log reduction) and Camp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV-irradiated cells did not affect PCR amplification, but long-amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. CONCLUSIONS: The long-amplicon PMA-PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture-based procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane-damaged cells, and also for inactivation mechanisms that cause direct DNA damage.
Authors: Kris M Weigel; Felicia K Nguyen; Moira R Kearney; John S Meschke; Gerard A Cangelosi Journal: Appl Environ Microbiol Date: 2017-05-01 Impact factor: 4.792