Willis Jessica R, Sivaganesan Mano, McMinn Brian, Korajkic Asja, Staley Christopher, Shanks Orin C
U.S. Environmental Protection Agency, Office of Research and Development, Center for Environmental Measurement and Modeling, Cincinnati, OH, 45268, USA.
Department of Surgery, University of Minnesota School, Minneapolis, MN, USA.
Environ Pollut. 2025 Oct 15;383:126806. doi: 10.1016/j.envpol.2025.126806. Epub 2025 Jul 9.
A recent study reports the genome sequence of ΦB124-14, a bacteriophage capable of infecting a narrow subset of Bacteroides spp. closely associated with the human gut. To investigate the use of ΦB124-14 for fecal source identification applications, the genome was interrogated for human-associated genetic regions using bioinformatic and laboratory approaches. Fifty-three primer sets were tested using 100 fecal samples from ten animal sources, primary influent sewage from 36 geographical locations across the United States, and environmental surface waters with known human sewage impact. Based on candidate primer set end-point PCR analyses and next generation amplicon sequencing, two novel quantitative real-time PCR assays (qPCR), PS28 and PS30, were designed and evaluated. Both qPCR assays exhibited a sensitivity of 86.1 %, a specificity of 100 %, and successfully detected ΦB124-14-like genetic markers in sewage impacted environmental water samples. PS28 and PS30 performance was then compared to established DNA-based viral (CPQ_056 and CPQ_064) and bacterial (HF183/BacR287 and HumM2) human-associated qPCR assays. Findings indicated ΦB124-14 bacteriophage-like qPCR assays exhibited superior specificity, but markers consistently occurred at lower concentrations in United States primary influent sewage. Reduced sensitivity may be insufficient for application in U.S. environmental waters, especially when trace levels of sewage are present. Furthermore, paired measurements of ΦB124-14 and crAssphage bacteriophage-like sequences in high volume (10 L) primary influent sewage samples (n = 38) indicated significant correlations ranging from r = 0.593 (p < 0.0001; PS30 versus CPQ_056) to r = 0.938 (p < 0.0001; PS28 versus PS30). A comparison of bacteriophage-like marker concentrations with cultured GB-124 in sewage samples showed no significant correlations (r ≤ 0.215, p ≥ 0.183).
最近的一项研究报告了噬菌体ΦB124 - 14的基因组序列，该噬菌体能够感染与人类肠道密切相关的一小部分拟杆菌属细菌。为了研究ΦB124 - 14在粪便来源鉴定应用中的用途，使用生物信息学和实验室方法对该基因组进行了人类相关基因区域的分析。使用来自十种动物来源的100份粪便样本、美国36个地理位置的主要进水污水以及已知受人类污水影响的环境地表水，测试了53组引物。基于候选引物组终点PCR分析和下一代扩增子测序，设计并评估了两种新型定量实时PCR检测方法（qPCR），即PS28和PS30。两种qPCR检测方法的灵敏度均为86.1%，特异性为100%，并成功检测到受污水影响的环境水样中的ΦB124 - 14样遗传标记。然后将PS28和PS30的性能与已建立的基于DNA的病毒（CPQ_056和CPQ_064）和细菌（HF183/BacR287和HumM2）人类相关qPCR检测方法进行比较。结果表明，ΦB124 - 14噬菌体样qPCR检测方法具有更高的特异性，但在美国主要进水污水中，这些标记物的浓度始终较低。灵敏度降低可能不足以应用于美国环境水域，尤其是当存在微量污水时。此外，对大量（10 L）主要进水污水样本（n = 38）中ΦB124 - 14和crAssphage噬菌体样序列的配对测量表明，相关性显著，范围从r = 0.593（p < 0.0001；PS30与CPQ_056）到r = 0.938（p < 0.0001；PS28与PS30）。污水样本中噬菌体样标记物浓度与培养的GB - 124的比较显示无显著相关性（r≤0.215，p≥0.183）。
