Polymerase chain reaction detection of nonviable bacterial pathogens

TLDR

PCR methods can detect pathogen nucleic acids in environmental samples, but amplification does not confirm organism viability. Our study shows PCR can detect nonviable cells when intact nucleic acids persist, but rapid degradation limits detection to a few weeks, indicating a risk of false positives and the need for careful sample storage and avoidance of filter sterilization for long-term preservation.

Abstract

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.