Abstract：
Microbial source tracking (MST) tools provide insights on fecal pollution levels in aquatic environments using predominantly quantitative PCR (qPCR) assays that target host-associated molecular marker genes. Existing wastewater-associated marker genes have shown limited or significant cross-reactions with non-human fecal samples. In this study, we mined the current Gut Phage Database (GPD) and designed a novel untreated wastewater-specific Bifidobacterium phage qPCR assay (i.e., Bifi assay). The sensitivity and specificity of the Bifi marker genes were assessed by collectively analyzing untreated (n = 33) and treated (n = 15) wastewater and non-human fecal samples (i.e., Rabbit, mouse, cow, horse, pig, chicken, sheep, dog, deer, kangaroos; n = 113) in Shenzhen, Guangdong Province, China and Brisbane, Australia. Bifi assay revealed 100% host-specificity against non-human fecal samples collected from Shenzhen and Brisbane. Furthermore, this marker gene was also detected in all untreated and treated wastewater samples, whose concentrations ranged from 5.54 to 6.83 log10 GC/L. In Shenzhen, the concentrations of Bifi marker gene were approximately two orders of magnitude lower than Bacteroides (HF183/BacR287 assay) and CrAssphage (CPQ_56 assay). The concentration of Bifi marker gene in untreated wastewater from Brisbane was 1.35 log10 greater than those in Shenzhen. Our results suggest that Bifi marker gene has the potential to detect and quantify the levels of human fecal pollution in Shenzhen and Brisbane. If additional detection sensitivity is required for environmental studies, Bifi marker gene should be paired with either CrAssphage or HF183/BacR287 marker genes.
摘要：
微生物来源追踪（MST）工具主要使用靶向宿主相关分子标记基因的定量PCR（qPCR）测定法，提供对水生环境中粪便污染水平的见解。现有的废水相关标记基因与非人类粪便样品显示出有限或显著的交叉反应。在这项研究中,我们挖掘了当前的肠道噬菌体数据库(GPD)，并设计了一种新型的未经处理的废水特异性双歧杆菌噬菌体qPCR检测方法(即，Bifi测定）。通过共同分析未处理（n=33）和处理（n=15）的废水和非人粪便样品（即，兔子,鼠标,母牛,马,猪,鸡肉,绵羊,狗,鹿,袋鼠；n=113)在深圳，广东省,中国和布里斯班，澳大利亚。Bifi测定显示针对从深圳和布里斯班收集的非人粪便样品的100%宿主特异性。此外，在所有未处理和处理过的废水样品中也检测到该标记基因，其浓度范围为5.54至6.83log10GC/L。在深圳,Bifi标记基因的浓度比拟杆菌（HF183/BacR287测定）和CrAssphage（CPQ_56测定）低约两个数量级。布里斯班未经处理的废水中Bifi标记基因的浓度比深圳高1.35log10。我们的结果表明，Bifi标记基因具有检测和量化深圳和布里斯班人类粪便污染水平的潜力。如果环境研究需要额外的检测灵敏度，Bifi标记基因应与CrAssphage或HF183/BacR287标记基因配对。
