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Rapid and Accurate Detection of SARS-CoV-2 Mutations using a Cas12a-based Sensing Platform
He Changsheng,Cailing Lin,Guosheng Mo,Binbin Xi,An′an Li,Dongchao Huang,Yanbin Wan,Feng Chen,Yufeng Liang,Qingxia Zuo,Wanqing Xu,Dongyan Feng,Guanting Zhang,Liya Han,Changwen Ke,Hongli Du,Lizhen Huang +16 more
TLDR
In this article, a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform was used to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G.Abstract:
The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-COV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of $10^{-17}$ M (approximately 6 copies/$\mu$L). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of $10^{-16}$ M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.read more
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Journal ArticleDOI
Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.
Bette T. Korber,Will Fischer,Sandrasegaram Gnanakaran,Hyejin Yoon,James Theiler,Werner Abfalterer,Nick Hengartner,Elena E. Giorgi,Tanmoy Bhattacharya,Brian T. Foley,Kathryn M. Hastie,Matthew Parker,David G Partridge,Cariad Evans,Timothy M. Freeman,Thushan I de Silva,Adrienne Angyal,Rebecca Brown,Laura Carrilero,Luke R. Green,Luke R. Green,Luke R. Green,Danielle C. Groves,Katie Johnson,Alexander J Keeley,Benjamin B Lindsey,Paul J. Parsons,Mohammad Raza,Sarah Rowland-Jones,Nikki Smith,Rachel Tucker,Dennis Wang,Matthew Wyles,Charlene McDanal,Lautaro G. Perez,Haili Tang,Alex Moon-Walker,Alex Moon-Walker,Alex Moon-Walker,Sean P. J. Whelan,Celia C. LaBranche,Erica Ollmann Saphire,David C. Montefiori +42 more
TL;DR: A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic, and it is found that the G614 variant grows to higher titer as pseudotyped virions.
Journal ArticleDOI
SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity
Lizhou Zhang,Cody B. Jackson,Huihui Mou,Amrita Ojha,Haiyong Peng,Brian D. Quinlan,Erumbi S. Rangarajan,Andi Pan,Abigail Vanderheiden,Mehul S. Suthar,Wenhui Li,Tina Izard,Christoph Rader,Michael Farzan,Hyeryun Choe +14 more
TL;DR: Pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614, and D614G may increase infectivity by assembling more functional S protein into the virion.
Journal ArticleDOI
The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay
Ralph Stadhouders,Suzan D. Pas,Jeer Anber,Jolanda J.C. Voermans,Ted H.M. Mes,Martin Schutten +5 more
TL;DR: The data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes, which should allow for more optimized development of real-time PCR assays involving primer- template mismatches.
Journal ArticleDOI
Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever.
Bai Jing,Haosi Lin,Li Haojian,Yang Zhou,Junshan Liu,Guorui Zhong,Luting Wu,Weifan Jiang,Hongli Du,Jinyi Yang,Qingmei Xie,Lizhen Huang +11 more
TL;DR: An RAA-Cas 12a-based system that combines recombinase aided amplification (RAA) and CRISPR/Cas12a for ASFV detection and uses CORDS to detect target DNA highly specifically using the lateral-flow strip readout, which displayed no cross-reactivity to other 13 swine viruses including classical swine fever (CSF).
Journal ArticleDOI
Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR
TL;DR: The ability to evaluate priming (and mispriming) rates and to predict their impacts provided a precise and quantitative description of assay performance and priming probabilities were found to be a good measure of analytical specificity.