Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.
Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.
复制标题
DOI：
10.1073/pnas.2010196117
复制
发表时间：
2020-10-13
影响因子：
11.1
通讯作者：
Collins JJ
中科院分区：
文献类型：
--
作者：
Lee RA;Puig H;Nguyen PQ;Angenent-Mari NM;Donghia NM;McGee JP;Dvorin JD;Klapperich CM;Pollock NR;Collins JJ
在无症状个体中检测亚微观疟疾是根除疟疾所必需的，在资源有限的情况下，这仍然是一个诊断空白。非恶性疟临床诊断是第二个缺口，因为这些感染的寄生虫密度较低，通常未被发现。我们描述了一种基于CRISPR的综合、60分钟、超灵敏和特异的诊断方法，用于可以填补这些空白的四种主要致病疟原虫物种。使用Sherlock(特异性高灵敏度酶报告解锁)平台，我们设计了检测限低于世界卫生组织推荐的检测限的方法。这些检测方法有一个简化的样品准备方法：夏洛克寄生虫快速提取方案，它省去了复杂的核酸提取步骤。我们的工作进一步将夏洛克平台转化为可现场部署的诊断工具。疟原虫的无症状携带者阻碍了疟疾的控制和根除。实现根除疟疾需要对低寄生虫密度感染(每微升血液100个寄生虫)进行超灵敏的诊断，这些感染在资源有限的环境下工作。非恶性疟疾的特点是感染密度较低，可能需要额外的治疗才能根治，因此也缺乏灵敏的临时点诊断。分子方法，如聚合酶链式反应，具有很高的敏感性和特异性，但仍然是高度复杂的技术，不适用于RLS。在这里，我们描述了一种基于CRISPR的诊断方法，用于利用核酸检测平台Sherlock(特定的高灵敏度酶报告解锁)对恶性疟原虫、间日疟原虫、卵形疟原虫和疟疾进行超灵敏的检测和区分。我们提出了一种简化的、现场适用的诊断方法，包括10分钟的Sherlock寄生虫快速提取方案，然后Sherlock通过荧光或横向流动条读数进行60分钟的疟疾物种特异性检测。我们用一种独立于核酸提取的简化样品制备方法优化了一锅冷冻干燥等温检测方法，并表明这些检测方法能够检测到每微升血液中两种寄生虫，这是世界卫生组织建议的检测极限。我们的恶性疟原虫和间日疟原虫检测对临床样本(5份恶性疟原虫和10份间日疟原虫样本)的敏感性和特异性均为100%。这项工作建立了一种现场适用的诊断方法，用于对无症状携带者的超敏感检测，以及对非恶性疟疾物种和低寄生虫密度恶性疟原虫感染的快速临床诊断。
Detection of submicroscopic malaria in asymptomatic individuals is needed for eradication and remains a diagnostic gap in resource-limited settings. Nonfalciparum clinical diagnostics are a second gap, as these infections have a low parasite density and are commonly undetected. We describe an integrated, 60-min, ultrasensitive and specific CRISPR-based diagnostic for the four major pathogenic Plasmodium species that can fill these gaps. Using the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform, we designed assays with limits of detection below that recommended by the World Health Organization. These assays have a simplified sample preparation method: the SHERLOCK parasite rapid extraction protocol, which eliminates complicated nucleic acid extraction steps. Our work further translates the SHERLOCK platform into a field-deployable diagnostic. Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.
登录
查看更多内容
影响因子：
5.7
作者：
Bourgard C;Albrecht L;Kayano ACAV;Sunnerhagen P;Costa FTM
通讯作者：
Costa FTM
影响因子：
28.1
作者：
Kaminski, Michael M.;Alcantar, Miguel A.;Collins, James J.
通讯作者：
Collins, James J.
影响因子：
4.7
作者：
Li, Linxian;Li, Shiyuan;Wang, Jin
通讯作者：
Wang, Jin
影响因子：
3.7
作者：
Gamboa D;Ho MF;Bendezu J;Torres K;Chiodini PL;Barnwell JW;Incardona S;Perkins M;Bell D;McCarthy J;Cheng Q
通讯作者：
Cheng Q
影响因子：
10.7
作者：
Katoh K;Standley DM
通讯作者：
Standley DM