Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi.
Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi.
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DOI：
10.1371/journal.pntd.0003884
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发表时间：
2015
影响因子：
3.8
通讯作者：
Ching WM
中科院分区：
文献类型：
--
作者：
Chao CC;Belinskaya T;Zhang Z;Ching WM
对恙虫病东方体（恙虫病东方体）和斑疹伤寒立克次体（斑疹伤寒立克次体）这两种分别是恙虫病和鼠斑疹伤寒的病原体进行灵敏、特异和快速的诊断检测是准确和及时诊断患者并确保他们得到适当治疗所必需的。采用横向流动试验（RPA-nfo）和实时荧光检测（RPA-exo），建立了针对恙虫病恙虫体47-kDa基因和斑疹伤寒恙虫体17 -kDa基因的重组酶聚合酶扩增（RPA）检测方法。RPA法检测恙虫病体和斑疹伤寒体的水平与定量PCR法相当。RPA-nfo和RPA-exo方法在检测伤寒致病菌17kda基因的敏感性方面相似。相反，RPA-exo对恙虫病体47 kDa基因的检测效果优于RPA-nfo。使用人类患者样本或感染小鼠样本评估恙虫病O. RPA检测的临床性能。10个PCR确认阳性样本中有8个经RPA检测为阳性，所有PCR确认阴性样本均经RPA检测为阴性。对带伤寒杆菌的患者血清也得到了类似的结果。该方法能够将恙虫病O.和伤寒R.与其他系统发育相关的细菌以及小鼠和人的DNA区分开来。此外，RPA-nfo反应在37℃下于20分钟内完成，然后在室温下孵育10分钟以形成免疫层析条。RPA-exo反应在39℃下20分钟内完成。采用防交叉污染盒检测RPA-nfo荧光扩增子提供了一种替代常规横向流动检测条的方法，后者更容易发生交叉污染。RPA检测为诊断恙虫病或鼠斑疹伤寒提供了一种高时效性、敏感性和特异性的替代方法。历史上，立克次体病原体是军事行动中发病和死亡的主要原因之一。最近，立克次体病在已知丰富的地区重新出现，或在未知存在的地区出现，对当地居民和旅行者构成重大的医疗问题。这些疾病很难诊断，因为它们往往与同一地理区域的许多其他疾病具有相似的症状。因此，临床医生提供及时准确的诊断尤其具有挑战性。利用重组酶聚合酶扩增（recombinase polymerase amplification， RPA）的核酸检测平台，建立了准确、灵敏、特异、易于操作的检测方法，分别检测恙虫病恙虫体和鼠伤寒恙虫病恙虫体。这些RPA分析提供了与qPCR相似的检测限和特异性。与qPCR不同，它们不需要热循环器，并提供可根据不同实验室能力修改的多个终点监测选项。这项工作提出了一种替代的检测平台，用于早期检测恙虫病或斑疹伤寒恙虫病感染，以便在设备齐全的实验室和资源有限的地区进行及时治疗。
Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus. Historically, rickettsial pathogens are among the leading causes of morbidity and mortality during military operations. Rickettsial diseases, lately, are reemerging in areas of known abundance or emerging in areas of unknown existence, posing a significant medical concern for local residents and travelers. The diseases are difficult to diagnose as they often share similar symptoms with many other diseases in the same geographical areas. Therefore, it is particularly challenging for clinicians to provide a timely and accurate diagnosis. A recombinase polymerase amplification (RPA)-based nucleic acid detection platform has been used to develop accurate, sensitive, specific, and easy-to-perform assays to detect O. tsutsugamushi or R. typhi, indicative of scrub typhus or murine typhus, respectively. These RPA assays provide similar limits of detection and specificity to that of qPCR. Unlike qPCR, they require no thermocycler and provide multiple end-point monitoring options amendable to different laboratory capabilities. This work presents an alternative assay platform for early detection of O. tsutsugamushi or R. typhi infection so that timely treatment can be prescribed in well-equipped laboratories as well as resource limited areas.
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7.4
作者：
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