Development of a Scorpion probe-based real-time PCR for the sensitive quantification of Bacteroides sp. ribosomal DNA from human and cattle origin and evaluation in spring water matrices.
Spring water from alpine catchments are important water resources but they can be vulnerable against faecal contamination. Potential faecal contamination sources are wildlife populations, pasturing activities, or alpine tourism. Unfortunately, no faecal source tracking method is available to date which is sensitive enough for appropriate spring water monitoring and source allocation. Our purpose was to develop a Duplex Scorpion real-time PCR approach for the specific and sensitive quantification of Bacteroides sp. 16S rDNA fragments from human and cattle origin. By the developed approach, detection of plasmids, carrying the respective biomarker sequence, was possible over a range of more than seven orders of magnitudes down to six copy numbers per PCR assay. Furthermore, the Duplex Scorpion real-time PCR allowed the specific quantification down to 50 targets in plasmid spiked spring water matrices. Results indicate that microbial source tracking appears feasible in spring water habitats by probe-based real-time PCR technologies. However, preliminary testing of the established approach on faecal samples collected from a representative alpine habitat did not allow unambiguous source allocation in all cases. In the future, the available sequence database has thus to be widened to allow reliable source tracking in alpine spring watersheds and even expand this approach to other potential faecal sources.
Stricker
A R
AR
Division of Applied Biochemistry and Gene Technology, Institute of Chemical Engineering, Technical University of Vienna, Getreidemarkt 9, 1060 Vienna, Austria.
Wilhartitz
I
I
Farnleitner
A H
AH
Mach
R L
RL
D000818:Animals; D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000032:analysis / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005618:Fresh Water / Q000382:microbiology*; D006801:Humans; D015336:Molecular Probe Techniques / Q000191:economics; D015335:Molecular Probes / Q000191:economics / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015335:Molecular Probes; D012336:RNA, Ribosomal, 16S