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COVID-19 Detection
At present, the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide on a large scale, threatening the global public health system and human life and health. Rapid, affordable and accurate diagnosis of SARS-CoV-2 is essential for early treatment and control of disease spread.
Overview of Coronavirus Detection Methods
Real-time fluorescence quantitative RT-PCR is currently the main clinical method for confirming the diagnosis of novel coronavirus pneumonia. The basic principle is to monitor the growth of the product amount in real time by fluorescently labeled specific probes and by tracking the labeling of the PCR product during the reaction, and to calculate the starting template amount based on the amplification curve. In addition, other methods such as digital PCR, macrogenome sequencing, and reverse transcription loop-mediated isothermal amplification (RT-LAMP) can also be used to detect neo-coronavirus.
CRISPR-Cas System-Based COVID-19 Detection Methods
In the past few years, CRISPR technology has shown great potential for molecular diagnosis due to its high sensitivity and specificity. In the ongoing COVID-19 pandemic, there is an increasing interest in implementing CRISPR-based diagnostic principles to develop rapid and accurate methods to detect SARS-CoV-2.
- CRISPR-Cas9 System-Based COVID-19 Detection
The ability of the CRISPR-Cas9 system to precisely identify and cleave specific DNA sequences means that it can be used for the development of novel molecular assays.
Researchers have developed a CRISPR-Cas9 technology-mediated three-line lateral flow strip assay platform and combined it with multiplex reverse transcription-recombinase polymerase isothermal amplification (RT-RPA) to successfully visualize two new coronavirus genes (Orf1ab and E gene) simultaneously and rapidly in a single test, in a single strip. The technology-mediated three-line lateral flow test strip platform features simple experimental conditions, fast detection, visualized reads, and comparable specificity and sensitivity to the gold standard RT-qPCR method. Therefore, this method can provide a new detection tool for the diagnosis of COVID-19.
- CRISPR-Cas12 System-Based COVID-19 Detection
The CRISPR-Cas12 system is an RNA-guided DNA-targeting CRISPR system. Scientists have discovered that LbCas12a has the remarkable ability to non-specifically cleave ssDNA upon binding to target DNA. Therefore, using this feature in combination with fluorescently labeled ssDNA signal reporter molecules, the DETECTR biosensing platform was developed for rapid and accurate detection of 2019-nCoV. Since then, various novel CRISPR/Cas12 biosensing platforms that can be used for neo-coronavirus detection have been developed, including biosensing systems such as HOLMES and HUDSON. By combining RPA, LAMP and other technologies, ultra-sensitive detection of neo-coronaviruses can be achieved.
Schematic of SARS-CoV-2 DETECTR Workflow (Broughton J.P. et al, Nat Biotechnol, 2020)
- CRISPR-Cas13 System-Based COVID-19 Detection
The CRISPR-Cas13 system is an RNA-directed RNA targeting system. Researchers have exploited the incidental cleavage activity of Cas13a to create the LwCas13a-based CRISPR/Cas biosensing system SHERLOCK, which has been successfully applied to the rapid detection of neo-coronaviruses. SHERLOCK assays work by recognizing an RNA sequence of approximately 20 bases, which is a unique base sequence for neo-coronaviruses. They bind to the target RNA sequence in solution by creating a "guide" RNA that is complementary to the target RNA sequence. When the "guide" RNA binds to the target RNA, the CRISPR tool's Cas13 "scissors" enzyme is activated, cutting off nearby single-stranded RNA. simultaneously, shearing releases individual fluorescent particles into the test solution, which glow when the sample is illuminated by a laser, indicating the presence of the virus.
Schematic of CRISPR-Cas13 System-Based SHERLOCK (Xie, S.Y. et al, Analytica Chimica Acta, 2021)
Our Services
Utilizing the DeteCasTM platform based on the CRISPR-Cas system, CD BioSciences can provide RNA detection service in COVID-19 detection. If you are interested in this service, please contact us.
References
- Gootenberg, J.S., et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017, 356(6336):438-442.
- Lin, X., et al. HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation. ACS Synthetic Biology. 2019.
- Broughton, J.P., et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020, 38: 870–874.
- Xie, S.Y., et al. Advancing sensing technology with CRISPR: From the detection of nucleic acids to a broad range of analytes-A review. Analytica Chimica Acta. 2021, 338848.
For research use only. Not intended for any clinical use.
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