Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology.
Bo Pang,Jingyang Xu,Yanming Liu,Hanyong Peng,Wei Feng,Yiren Cao,Jinjun Wu,Huyan Xiao,Kanti Pabbaraju,Graham Tipples,Michael A. Joyce,Holly A. Saffran,D. Lorne Tyrrell,Hongquan Zhang,X. Chris Le +14 more
TLDR
In this paper, a single-tube assay for SARS-CoV-2 in patient samples was developed, which combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a.Abstract:
We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.read more
Citations
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CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules
Wei Feng,Ashley M. Newbigging,Jeffrey Tao,Yiren Cao,Hanyong Peng,Connie Le,Jinjun Wu,Bo Pang,Bo Pang,Juan Li,D. Lorne Tyrrell,Hongquan Zhang,X. Chris Le +12 more
TL;DR: Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.
Journal ArticleDOI
An electrochemical biosensor for sensitive analysis of the SARS-CoV-2 RNA.
TL;DR: In this article, the authors proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA in the range of 0.1-1000 pM with the detection limit as low as 26 fM.
Journal ArticleDOI
Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP.
Yaqin Zhang,Minyan Chen,Chengrong Liu,Jiaqi Chen,Xinyi Luo,Yingying Xue,Qiming Liang,Qiming Liang,Li Zhou,Yu Tao,Mingqiang Li,Di Wang,Di Wang,Jianhua Zhou,Jiasi Wang +14 more
TL;DR: In this article, a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LA MP/AuN P (CLAP) was developed for rapid and sensitive detection of SARS-CoV-2.
Journal ArticleDOI
CRISPR-Cas based virus detection: Recent advances and perspectives.
TL;DR: In this article, the authors reviewed the recent advances in virus detection with CRISPR-Cas systems, especially CRISpl-Cas12a/Cas13a, characterized by their sensitivity, specificity, high base resolution and programmability upon nucleic acid recognition.
Journal ArticleDOI
Review of COVID-19 testing and diagnostic methods
Olena Filchakova,Dina Dossym,Aisha Ilyas,T. K. Kuanysheva,Altynay Abdizhamil,Rostislav Bukasov +5 more
TL;DR: More than six billion tests for COVID-19 have been already performed in the world as discussed by the authors , and even minimal improvement in any of them may have noticeable impact on life in the many countries of the world.
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