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

Abstract

Aims: To validate host-specific Bacteroidales assays to identify faecal-sourcecontamination of drinking water sources in the Kathmandu Valley, Nepal.Methods and Results: A total of 54 composite faecal-source samples werecollected 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 bestperforming human-specific, ruminant-specific and pig-specific assaysrespectively. The BacCan SYBR assay highly cross-reacted with other species,resulting in poor performance. Furthermore, these validated assays wereapplied to microbial source tracking (MST) of 74 drinking water samples. Outof these, 20, 12 and 4% samples were judged contaminated by human,ruminant and pig faeces respectively. Detection ratios of human and ruminantfaecal markers were relatively higher in built-up and agricultural areasrespectively.Conclusion: BacHum, BacR and Pig2Bac assays were found suitable for MSTand both, human and animal faecal contaminations of drinking water sourceswere common in the valley.Significance and Impact of the study: MST could be an effective tool forpreparing the faecal pollution strategies as these are site specific.