Development of on-site rapid diagnostic test for early detection of COVID-19 based on CRISPRCas and Surface-Enhanced Raman Spectroscopy

A: Background During unpredicted epidemic outbreaks such as COVID-19, establishment of early-stage diagnostic tools, which can favor timely diagnosis, and control the spread of the disease in order to sustain and monitor people's lives are always a priority in personalized medicine. Most employed tools such as real-time PCR (Polymerase chain reaction) and ELISA (Enzyme linked immunosorbent assay)-based techniques are still expensive, tedious and their sensitivity is of concern. CRISPR/Cas systems (clustered regularly interspaced short palindromic repeats)/ associated Cas proteins), hold promise in the development of novel and reliable diagnostic tools. CRISPR/Cas systems present the potential to turn into the next-generation diagnostic tools, owing to its high sensitivity, and specificity since they can be multiplexed and reach SNPs (Single nucleotide polymorphisms) detection. B: Goals and Objectives The project aims to develop a high fidelity CRISPR kit for COVID-19 nucleic acid detection based on CRISPR-Cas12a and a probe-based lateral flow biosensor or handheld SERS system. The design uses: • use CRISPR Cas12a/b as the recognition system • Reverse transcriptase Loop-mediated isothermal amplification (RT-LAMP) as the signal amplification technique, and • Lateral flow strip and/or fluorescence reader or portable SurfaceEnhanced Raman Spectroscopy will be used for results readout. C: Methods The research project considers the following methodology approaches: i. Clinical evaluation of COVID-19 and impact assessment: Study the main content and key scientific and technical issues to be solved • Site selection and data collection: data available for lab analysis • Laboratory analysis: Identification of COVID19 • Data modeling and interpretation: Mapping of the disease status in the country • Manuscript writing and submission: Manuscript accepted for publication ii. Reverse transcription Loop-mediated isothermal amplification: Lab design, development and testing of the diagnostic test device kit • Primer design and synthesis: design of reporter molecule and capture probe (designed primers ordered using specific software) • Preliminary establishment of the RT-LAMP detection system: RT-LAMP system design and software modelling • Optimization of the RT-LAMP reaction system, Sensitivity test of the RT-LAMP detection method and Optimization of the RT-LAMP reaction system • CRISPR/Cas12 reaction design: Model designed and tested • Design of CIALFB onepot system: The device is operationally tested and protocol optimization (High sensitive and specific biosensor chip) iii. Test performance: Application of COVID-19 Rapid Diagnostic Test - On field testing of accuracy, sensitivity, specificity • Testing the kit on samples: COVID-19 detection in sub-clinical samples (with Wider pathogenic applicability) • Results comparison (with gold standard methods): Rapid vs. classic wet lab: Sensitivity, specificity and accuracy tested (with High sensitivity, specificity and accuracy) D: Expected outcomes Expected research results include: • Product of COVID-19 nucleic acid rapid test kit and a handheld SERS detector: developed CRISPR-based lateral flow biosensor for the detection of COVID-19 nucleic acid for on-site detection. This is a quick, easy and cheap Covid-19 detection kit with high sensitivity ( the detection limit can reach a level (10−18M), which is equal to a single microbial gene copy detection), high specificity (LAMP coupled with the specific recognition of gRNA ensures the ultra-high target detection), and Broad applicability: Freeze-dried products, easy to transport at room temperature • Application for one related invention patent • Publish one high-level scientific paper