The analysis of host-associated microorganisms by quantitative polymerase chain reaction (qPCR) has long been widely applied as a microbial source tracking (MST) tool for identifying the sources of fecal contamination. Although numerous studies have been conducted across Asia, relevant research on Indonesia is scarce. In this study, we evaluated the performance of various human- and animal-specific MST markers and applied the results to the assessment of the water quality of a river in West Java, Indonesia. Fecal samples from seven animal species (n = 43), sewage samples (n = 7), and river water samples (n = 48) were collected from eight locations along a river in West Java from August to September 2023 (dry season) and February to March 2024 (wet season). Among the 16 host-specific MST markers tested using qPCR, the detection ratio of human-specific markers in the target samples showed considerable variation (43 %-100 %). Ruminant-specific markers, including bacterial (BacCow and BacR) and mitochondrial DNA (Bovine-mtDNA) markers, demonstrated a 100 % detection ratio in their target samples. In the nontarget samples, the positive ratio of MST markers varied widely from 0 % (AiV-1) to 81 % (BacCow). crAssphage and Bovine-mtDNA were identified as the most effective MST markers specific to humans and ruminants, respectively. pBI143, as a newly established human-specific marker, demonstrated commendable performance with 100 % sensitivity and 77 % specificity. This study confirmed the presence of MST markers in the river water, with a positive ratio of 38 % for Bovine-mtDNA and 67 % for crAssphage during the dry season; the values increased during the wet season to 54 % and 88 %, respectively. The results emphasize the importance of using several types of MST markers to obtain comprehensive results and insight into contamination patterns, as well as the necessity for further research across diverse sub-watersheds and other water sources.

With increasing contamination in aquatic ecosystems, effective monitoring is crucial to preserve biodiversity and protect public health. This study quantified bioindicators (red swamp crayfish (Pcla), Genji-firefly (Lcr2), Ayu fish (Paa), and caddisfly (Sma)), microbial source tracking markers (ruminants (BacR), pigs (Pig2Bac), and humans (gyrB)), and a fecal indicator bacterium (Escherichia coli (sfmD)) using quantitative PCR on river water samples collected every 2 h between 21 and 22 July 2023 (from the Omo and Bingushi Rivers in Yamanashi Prefecture, Japan). Initially, the optimal filter sizes of 1.0, 0.65, and 0.22 µm were evaluated, where the 0.65 µm filter yielded higher Paa concentrations (Kruskal–Wallis test, p < 0.05) and was used subsequently. BacR and Paa exhibited 100% detection in the Omo (13/13) and Bingushi (13/13) Rivers with concentrations of 5.0 log₁₀ and 5.5 log₁₀ copies/L, respectively. These concentrations were used to assess 24 h temporal variability, but no significant fluctuations or cyclical trends between morning, afternoon, evening, and night were observed in either river. The BacR–Paa pair exhibited perfect positive detection correlation (Φ = 1.0) and complete similarity (Jaccard Index = 1.0), but a moderate negative correlation of mean concentrations highlights the importance of considering habitat overlaps and behavioral synchronicity.