A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area.
OBJECTIVE
The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes.


RESULTS
The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence.


CONCLUSIONS
The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water.


CONCLUSIONS
The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.
Reischer
G H
GH
Institute for Chemical Engineering, Gene Technology Group, Vienna University of Technology, Vienna, Austria.
Kasper
D C
DC
Steinborn
R
R
Farnleitner
A H
AH
Mach
R L
RL
D000818:Animals; D001317:Austria; D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014876:Water Pollution
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S