Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa
Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa
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DOI：
10.1021/acs.analchem.5b03267
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发表时间：
2016-02-02
影响因子：
7.4
通讯作者：
Richards-Kortum, Rebecca
中科院分区：
文献类型：
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作者：
Crannell, Zachary;Castellanos-Gonzalez, Alejandro;Richards-Kortum, Rebecca
这项工作描述了一种具有横向流动读数的多重重组酶聚合酶扩增（RPA）试验，该试验能够同时检测和区分任何引起腹泻的原生动物贾第鞭毛虫、隐孢子虫和内阿米巴原虫的DNA。这些寄生虫加在一起，对全球腹泻疾病负担造成重大影响。这些寄生虫的鉴别诊断传统上是通过粪便显微镜完成的。然而，显微镜是不敏感的，可以错过多达一半的病例。基于dna的诊断，如聚合酶链反应（PCR），要灵敏得多；然而，它们依赖于昂贵的热循环设备，限制了它们在集中参考实验室的可用性。等温DNA扩增平台，如本研究中使用的RPA平台，减轻了对热循环设备的需求，并有可能扩大更敏感的诊断途径。到目前为止，还没有开发出能够同时检测和区分由不同病原体引起的感染的多重RPA测定方法。我们开发了一种多重RPA检测方法来检测贾第鞭毛虫、隐孢子虫和内阿米巴原虫DNA的存在。多重检测使用合成DNA进行特征化，其中计算出的检测限分别为合成贾第鞭毛虫、隐孢子虫和内阿米巴虫靶标每次反应的403、425和368个基因拷贝（大约比单一RPA检测中相同靶标高1.5个数量级）。多重检测还利用从粪便样本中提取的活寄生虫的DNA进行鉴定，计算出贾第鞭毛虫、隐孢子虫和内阿米巴原虫每次反应的检出限分别为病、6和9种寄生虫。这种概念验证分析可以通过重新设计引物和探针序列来重新配置以检测各种各样的目标。
This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be ill, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.