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Source Tracking based on Microbial Communities: Sample Collection and ddPCR/qPCR
Author(s) -
Orellana Marilyn,
Veenstra Brendan,
Veine Cameryn,
Vollmert Grace,
Pikaart Michael
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r2949
Subject(s) - bacteroidetes , biology , taqman , source tracking , 16s ribosomal rna , bacteria , contamination , context (archaeology) , primer (cosmetics) , fecal coliform , microbiology and biotechnology , real time polymerase chain reaction , chemistry , genetics , ecology , gene , water quality , paleontology , organic chemistry , world wide web , computer science
Fecal contamination serves as a major public health threat, especially in drinking or recreational waters. This is particularly concerning because it promotes the exposure of communities to harmful pathogenic bacteria. Identifying the source of contamination serves as an important context when mitigating the effects of the pollution or preventing it from initially entering the water. Microbial source tracking (MST) is employed to achieve the quantification of dominant sources of fecal contamination. Specifically in this study, a library‐independent method was utilized in the sample level detection of host‐associated 16s rRNA markers. Among the most prevalent bacteria in human and animal gut microbiomes is the phylum Bacteroidetes, which prove to be extremely useful in MST assays. TaqMan primers and probes targeting host‐independent (universal) Bacteroidetes (BacUni), human‐host specific (HF183), and pig host specific (Pig2Bac) were utilized. Dye‐based SYBR assays targeting avian bacteria Helicobacter (GFD) were also investigated. Solid fecal samples were acquired from a local petting zoo and surrounding beach sites. Water samples from various lakes, streams, and wastewater treatment plants were also obtained. Samples were filtered using membrane filtration technology and DNA was isolated using DNEasy PowerSoil kit. Upon DNA isolation, the concentration of DNA was measured using NanoDrop and the samples were subjected to qPCR or ddPCR assays. Results indicate effective primer/probe selectivity. Cross‐reactivity between samples and primers demonstrated that the targets were not exclusive to the presumed host and could overlap. Future research includes testing primers on samples from a wider range of animals and water sources. Additionally, a comparison of qPCR results to those from ddPCR and quantifying any differences within or between the two.