Validation and application of high-throughput quantitative PCR for the simultaneous detection of microbial source tracking markers in environmental water.

ABSTRACT

No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a high-throughput quantitative polymerase chain reaction (HT-qPCR) method for the simultaneous detection of multiple MST markers. A total of 26 fecal-source samples that had been previously collected from human sewage (n = 6) and ruminant (n = 3), dog (n = 6), pig (n = 6), chicken (n = 3), and duck (n = 2) feces in the Kathmandu Valley, Nepal, were used to validate 10 host-specific MST markers, i.e., Bacteroidales (BacHum, gyrB, BacR, and Pig2Bac), mitochondrial DNA (mtDNA) (swine, bovine, and Dog-mtDNA), and viral (human adenovirus, porcine adenovirus, and chicken/turkey parvovirus) markers, via HT-qPCR. Only Dog-mtDNA showed 100 % accuracy. All the tested bacterial markers showed a sensitivity of 100 %. Nine of the 10 markers were further used to identify fecal contamination in groundwater sources (n = 54), tanker filling stations (n = 14), drinking water treatment plants (n = 5), and river water samples (n = 6). The human-specific Bacteroidales marker BacHum and ruminant-specific Bacteroidales marker BacR was detected at a high ratio in river water samples (83 % and 100 %, respectively). The results of HT-qPCR were in agreement with the standard qPCR. The comparable performances of HT-qPCR and standard qPCR as well as the successful detection of MST markers in the fecal-source and water samples demonstrated the potential applicability of these markers for detecting fecal contamination sources via HT-qPCR.