Book Description
ABSTRACT: Polymerase chain reaction (PCR) methods to detect indicators of fecal contamination are more rapid and specific than current government-recommended culture-based methods. However, PCR does not distinguish among live cells, dead cells or extracellular DNA. Propidium monoazide (PMA) is a DNA-binding dye that only permeates membrane-compromised (dead) cells. Once inside a dead cell, it binds DNA, preventing subsequent PCR amplification, allowing for PCR detection of only live cells. I used PMA coupled with quantitative PCR (qPCR) to compare persistence of live human-associated Bacteroidetes (HB) and Methanobrevibacter smithii to dead HB and M. smithii and their extracellular DNA. The HB qPCR signal was detected for 5 to 6 days (d) in mesocosms and was unaffected by PMA treatment. Thus, HB DNA, detected by PCR, was due to live bacteria and would indicate recent human fecal contamination if present in natural water. In contrast, the M. smithii qPCR signal persisted the entire study without PMA treatment but only 5 to 6 d with PMA treatment. Thus, although live M. smithii decreased in the mesocosms, its DNA persisted. Thus, M. smithii DNA, as detected by PCR, could be due to live cells, dead cells or extracellular DNA, leading to false indication of fecal contamination.
