Specificity and sensitivity evaluation of novel and existing Bacteroidales and Bifidobacteria-specific PCR assays on feces and sewage samples and their application for microbial source tracking in Ireland.
Three novel ruminant-specific PCR assays, an existing ruminant-specific PCR assay and five existing human-specific PCR assays, which target 16S rDNA from Bacteroidales or Bifidobacteria, were evaluated. The assays were tested on DNA extracted from ruminant (n=74), human (n=59) and non-ruminant animal (n=44) sewage/fecal samples collected in Ireland. The three novel PCR assays compared favourably to the existing ruminant-specific assay, exhibiting sensitivities of 91-100% and specificities of 95-100% as compared to a sensitivity of 95% and specificity of 94%, for the existing ruminant-specific assay. Of the five human-specific PCR assays, the assay targeting the Bifidobacterium catenulatum group was the most promising, exhibiting a sensitivity of 100% (with human sewage samples) and a specificity of 87%. When tested on rural water samples that were naturally contaminated by ruminant feces, the three novel PCR assays tested positive with a much greater percentage (52-87%) of samples than the existing ruminant-specific assay (17%). These novel ruminant-specific assays show promise for microbial source tracking and merit further field testing and specificity evaluation.
Environmental Microbiology Research Unit, Microbiology, School of Natural Sciences, National University of Ireland, Galway, University Road, Galway, Ireland. siobhan.dorairaj@nuigalway.ie
Siobhán
S
Dorai-Raj
Justin
J
O' Grady
Emer
E
Colleran
D041963:Bacteroidetes / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001644:Bifidobacterium / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D007494:Ireland; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D012722:Sewage / Q000382:microbiology*; D014873:Water Pollutants / Q000302:isolation & purification*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D014873:Water Pollutants