Validation of host-specific Bacteroidales quantitative PCR assays and their application to microbial source tracking of drinking water sources in the Kathmandu Valley, Nepal

Aims: To validate host-specific Bacteroidales assays to identify faecal-source contamination of drinking water sources in the Kathmandu Valley, Nepal. Methods and Results: A total of 54 composite faecal-source samples were collected from human sewage, ruminants, pigs, dogs, chickens and ducks, which were analysed by quantitative polymerase chain reaction using human-specific (BacHum, HF183 SYBR, gyrB and HF183 TaqMan), ruminant-specific (BacCow and BacR), pig-specific (Pig2Bac and PF163) and dog-specific assays (BacCan SYBR). The BacHum, BacR and Pig2Bac assays were judged the best performing human-specific, ruminant-specific and pig-specific assays respectively. The BacCan SYBR assay highly cross-reacted with other species, resulting in poor performance. Furthermore, these validated assays were applied to microbial source tracking (MST) of 74 drinking water samples. Out of these, 20, 12 and 4% samples were judged contaminated by human, ruminant and pig faeces respectively. Detection ratios of human and ruminant faecal markers were relatively higher in built-up and agricultural areas respectively. Conclusion: BacHum, BacR and Pig2Bac assays were found suitable for MST and both, human and animal faecal contaminations of drinking water sources were common in the valley. Significance and Impact of the study: MST could be an effective tool for preparing the faecal pollution strategies as these are site specific.