Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment
Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment
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DOI：
10.1016/j.watres.2014.12.008
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发表时间：
2015-03-01
期刊：
影响因子：
12.8
通讯作者：
Griffith, John F.
中科院分区：
文献类型：
--
作者：
Cao, Yiping;Raith, Meredith R.;Griffith, John F.
尽管广泛应用于海滩水质监测和微生物来源鉴定，但定量PCR（qPCR）方法产生的结果受到依赖定量标准引入的偏倚的影响。数字PCR技术提供了直接的、无标准的定量，并且可能潜在地减轻或大大减少其他qPCR限制，例如难以多重化和对PCR抑制的敏感性。本研究检查了采用双重液滴数字PCR（ddPCR）测定同时定量肠球菌属的有效性。和人粪便相关的HF183标记物用于水质评价。使用参比物质和131份粪便和水样，与qPCR和单纯ddPCR并行评价了双重ddPCR的性能。粪便和水样品的结果在ddPCR和单纯qPCR之间高度相关（系数> 0.93，p < 0.001）。在qPCR中双链肠球菌和HF183导致竞争，并导致相对于另一个低浓度的靶标的未检测或低估，而通过单重和双链ddPCR产生的结果是一致的，并且通常彼此无法区分。ddPCR显示出更大的抑制耐受性，在比qPCR耐受的抑制剂浓度高一到两个数量级的抑制剂浓度下，对定量没有明显影响。总体而言，ddPCR还表现出更高的精确度，更高的运行间重复性，类似的诊断灵敏度和特异性的HF 183标志物，但较低的定量上限比qPCR。数字PCR有可能成为qPCR的可靠和经济的替代品，用于娱乐用水监测和粪便源识别。这项研究的发现也可能对水研究的其他方面感兴趣，例如病原体和抗生素抗性基因的检测。(C)2014爱思唯尔有限公司版权所有。
Despite wide application to beach water monitoring and microbial source identification, results produced by quantitative PCR (qPCR) methods are subject to bias introduced by reliance on quantitative standards. Digital PCR technology provides direct, standards-free quantification and may potentially alleviate or greatly reduce other qPCR limitations such as difficulty in multiplexing and susceptibility to PCR inhibition. This study examined the efficacy of employing a duplex droplet digital PCR (ddPCR) assay that simultaneously quantifies Enterococcus spp. and the human fecal-associated HF183 marker for water quality assessment. Duplex ddPCR performance was evaluated side-by-side with qPCR and simplex ddPCR using reference material and 131 fecal and water samples. Results for fecal and water samples were highly correlated between ddPCR and simplex qPCR (co-efficients > 0.93, p < 0.001). Duplexing Enterococcus and HF183 in qPCR led to competition and resulted in non-detection or underestimation of the target with low concentration relative to the other, while results produced by simplex and duplex ddPCR were consistent and often indistinguishable from one another. ddPCR showed greater tolerance for inhibition, with no discernable effect on quantification at inhibitor concentrations one to two orders of magnitude higher than that tolerated by qPCR. Overall, ddPCR also exhibited improved precision, higher run-to-run repeatability, similar diagnostic sensitivity and specificity on the HF183 marker, but a lower upper limit of quantification than qPCR. Digital PCR has the potential to become a reliable and economical alternative to qPCR for recreational water monitoring and fecal source identification. Findings from this study may also be of interest to other aspects of water research such as detection of pathogens and antibiotic resistance genes. (C) 2014 Elsevier Ltd. All rights reserved.