Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India

Mitsunori Odagiri; Alexander Schriewer; Kaitlyn Hanley; Stefan Wuertz; Pravas R Misra; Pinaki Panigrahi; Marion W Jenkins

doi:10.1016/j.scitotenv.2014.09.040

Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India

Sci Total Environ. 2015 Jan 1:502:462-70. doi: 10.1016/j.scitotenv.2014.09.040. Epub 2014 Oct 3.

Authors

Mitsunori Odagiri¹, Alexander Schriewer¹, Kaitlyn Hanley¹, Stefan Wuertz², Pravas R Misra³, Pinaki Panigrahi⁴, Marion W Jenkins⁵

Affiliations

¹ Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
² Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; Singapore Centre on Environmental Life Sciences Engineering (SCELSE), School of Biological Sciences, and School of Civil and Environmental Engineering, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. Electronic address: swuertz@ucdavis.edu.
³ Asian Institute of Public Health, Bhubaneswar, Odisha, India.
⁴ Departments of Epidemiology and Pediatrics, Center for Global Health and Development, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198, USA.
⁵ Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

PMID: 25285421
DOI: 10.1016/j.scitotenv.2014.09.040

Abstract

We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India.

Keywords: Bacteroidales; Fecal pollution; India; Microbial source tracking; Quantitative PCR.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

Animals
Bacteroidetes / classification
Bacteroidetes / genetics*
Environmental Monitoring / methods*
Feces / microbiology*
Humans
India
Real-Time Polymerase Chain Reaction
Sewage / microbiology*

Substances

Sewage