AWWA WQTC64072

Given the rapid progress in molecular technique development and the discussion aboutthe discrepancy between culturability and viability, there is increasing interest in knowingwhether the use of plate counts for water safety monitoring could be replaced or at leastsupplemented with more rapid real-time molecular assays. The potential techniquesinclude quantitative PCR or DNA-chips for detection and enumeration of specificpathogens or indicator organisms. However, one of the major drawbacks of DNA-baseddiagnostics is its inability to discriminate between live and dead cells. Due to thepersistence of DNA in the environment after cells have lost their viability, DNA-basedassays can lead to an overestimation of the pathogenic risk as signals can originate fromboth live and dead cells. This paper presents the treatment of bacterial samples with thenovel chemical, propidium monoazide (PMA), which allows for the selective suppressionof the signal from dead cells based on membrane integrity. PMA can only penetrate deadcells with permeabilized cell membranes. Upon intercalation into the DNA, lightexposure results in covalent crosslinkage of PMA to the DNA. This modification stronglyinhibits PCR amplification and limits molecular diagnostics to those cells with intactmembranes which prevented PMA from entering the cytoplasm and consequently do nothave PMA bound to their DNA. This method was used to monitor the disinfectionefficacy of model pathogens using hypochlorite, benzalkonium chloride and UV. Includes 13 references.

Product Details

Edition:

Vol. - No.

Published:

11/01/2006

Number of Pages:

6

File Size:

1 file , 320 KB