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Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (\u3ci\u3eGrus canadensis\u3c/i\u3e) Fecal Pollution

机译:定量pCR分析靶向的开发与评价 沙丘鹤(\ u3ci \ u3eGrus canadensis \ u3c / i \ u3e)粪便污染

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摘要

While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n=553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n=16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n=165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.
机译:尽管普拉特河中的微生物水质受季节性迁移的起重机排泄物的影响,但目前尚无研究起重机粪便污染的方法。在这里,我们使用16S rRNA基因粪便克隆文库的系统发育分析来分析起重机粪便中的微生物种群。使用这些序列,开发了一种新型的起重机定量PCR(Crane1)分析,并通过确定其在环境水中的宿主特异性和检测能力来评估其作为微生物来源跟踪(MST)分析的适用性。鹤类排泄物中的细菌主要由杆菌和变形杆菌组成,与拟杆菌和梭状芽胞杆菌同源的序列非常少。 Crane1标记物靶向未分类的乳杆菌序列的优势进化枝,该序列与滨海卡特氏菌密切相关。 Crane1标记的宿主分布相对较高,在所测试的起重机粪便样本中占69%(66/96)呈阳性。该测定还显示出高的宿主特异性,其中95%的非靶标粪便样品(即,n = 553; 20种不同的自由程宿主)为阴性。在假定的受起重机影响的水样中(n = 16),Crane1分析的阳性率为88%,而不受起重机影响的水样中没有一个为阳性(n = 165)。 Crane1 MST标记的贝叶斯统计模型表明,从环境水样中检测出真阳性信号的可信度很高,而假阴性信号的概率也很小。总之,这些数据表明,新开发的标记物可用于环境监测研究中,以研究起重机粪便的污染动态。

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