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Rapid, accurate, nucleobase detection using FnCas9
medRxiv - Genetic and Genomic Medicine Pub Date : 2020-09-14 , DOI: 10.1101/2020.09.13.20193581
Mohd. Azhar , Rhythm Phutela , Manoj Kumar , Asgar Hussain Ansari , Riya Rauthan , Sneha Gulati , Namrata Sharma , Dipanjali Sinha , Saumya Sharma , Sunaina Singh , Sundaram Acharya , Deepanjan Paul , Poorti Kathpalia , Meghali Aich , Paras Sehgal , Gyan Ranjan , Rahul C. Bhoyar , Khushboo Singhal , Harsha Lad , Pradeep Kumar Patra , Govind Makharia , Giriraj Ratan Chandak , Bala Pesala , Debojyoti Chakraborty , Souvik Maiti ,

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.

中文翻译:

使用FnCas9进行快速,准确的核碱基检测

通过即时诊断方法快速检测DNA和RNA中的病原体序列或变体,对于加速临床预后非常有价值,正如最近在COVID-19爆发中所见证的那样。依赖qPCR或测序的传统方法很难在资源有限的环境中实施,因此需要开发出性能强大的精确替代测试策略。在这里,我们介绍了FnCas9编辑器链接均匀检测测定(FELUDA),它采用基于Cas9的直接酶读数来检测核苷酸序列和鉴定核碱基同一性,而无需报道分子的反式裂解活性。我们证明FELUDA在检测包括突变的杂合子的单核苷酸变体(SNV)方面​​是100%准确的,并以网络工具JATAYU的形式提供了一种简单的设计策略以供实施。FELUDA是半定量的,可以适应多种信号检测平台,可以快速设计和部署,以用于诸如传染病暴发(如COVID-19)之类的多功能应用。使用1小时内的侧向流量读数,FELUDA在临床样品的所有病毒载量范围内均显示出100%的敏感性和97%的特异性。结合RT-RPA和通过条带评估(TOPSE)预测的智能手机应用程序“真实结果”,我们提供了FELUDA的原型,用于在家中检测CoV-2。可以适应多种信号检测平台,并且可以快速设计和部署,以用于多种应用,例如传染病暴发(如COVID-19)。使用1小时内的侧向流量读数,FELUDA在临床样品的所有病毒载量范围内均显示出100%的敏感性和97%的特异性。结合RT-RPA和通过条带评估(TOPSE)预测的智能手机应用真实结果,我们提供了FELUDA的原型,用于在家中检测CoV-2。可以适应多种信号检测平台,并且可以快速设计和部署,以用于多种应用,例如传染病暴发(如COVID-19)。使用1小时内的侧向流量读数,FELUDA在临床样品的所有病毒载量范围内均显示出100%的敏感性和97%的特异性。结合RT-RPA和通过条带评估(TOPSE)预测的智能手机应用程序“真实结果”,我们提供了FELUDA的原型,用于在家中检测CoV-2。
更新日期:2020-09-14
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