ArticleACS synthetic biology2022
Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments.
Article in ACS synthetic biology, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 67 citations in OpenAlex.
- Article
- Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a.Nucleic acids research · 2026Article
- Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection.Nucleic acids research · 2026Article
- CRISPR-based point-of-care diagnostics for viral hepatitis: from molecular design to clinical implementation.Frontiers in bioengineering and biotechnology · 2026Review
- An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing ofJournal of clinical microbiology · 2025Article
- A One-Pot RT-LAMP Diagnostic Assay for SARS-CoV‑2 from Saliva Samples.ACS measurement science au · 2025Article
- One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and Perspectives.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Review
- A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean.Scientific reports · 2025Article
- Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.ACS synthetic biology · 2025Review
- Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.Nature communications · 2025Article
- OT-Mation: an open-source code for parsing CSV files into Python scripts for control of OT-2 liquid-handling robotics.Synthetic biology (Oxford, England) · 2025Article
- CRISPR-based strategies for sample-to-answer monkeypox detection: current status and emerging opportunities.Nanotechnology · 2024Review
- Affinity molecular assay for detecting Candida albicans using chitin affinity and RPA-CRISPR/Cas12a.Nature communications · 2024Article
- Evolution and Impact of Nucleic Acid Amplification Test (NAAT) for Diagnosis of Coronavirus Disease.Analytical chemistry · 2024Review
- Optimizing Nanosuspension Drug Release and Wound Healing Using a Design of Experiments Approach: Improving the Drug Delivery Potential of NDH-4338 for Treating Chemical Burns.Pharmaceutics · 2024Article
- CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.Biosensors · 2023Article
- Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.BMC microbiology · 2023Article
- CRISPR-Cas-based techniques for pathogen detection: Retrospect, recent advances, and future perspectives.Journal of advanced research · 2023Review
- CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA.Analytica chimica acta · 2023Article
- Nanotechnology-based diagnostic methods for coronavirus: From nucleic acid extraction to amplification.Biosensors & bioelectronics: X · 2023Review
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.
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