title: Validation and field testing of library-independent microbial source tracking methods in the Gulf of Mexico.
abstract: Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts' workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.
authors: University of South Florida, Department of Integrative Biology SCA110, 4202 E. Fowler, Tampa, FL 33620, USA. vharwood@cas.usf.edu
authors: Valerie J
authors: VJ
authors: Harwood
authors: Miriam
authors: M
authors: Brownell
authors: Shiao
authors: S
authors: Wang
authors: Joe
authors: J
authors: Lepo
authors: R D
authors: RD
authors: Ellender
authors: Abidemi
authors: A
authors: Ajidahun
authors: Kristen N
authors: KN
authors: Hellein
authors: Elizabeth
authors: E
authors: Kennedy
authors: Xunyan
authors: X
authors: Ye
authors: Christopher
authors: C
authors: Flood
mesh_terms: D001267:Atlantic Ocean; D041963:Bacteroidetes / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods* / Q000592:standards; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D041581:Methanobrevibacter / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012623:Seawater / Q000382:microbiology*; D012722:Sewage / Q000382:microbiology; D014873:Water Pollutants / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D012722:Sewage; D014873:Water Pollutants