Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. The clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei was reported for the detection of SARS-CoV-2. 154 nasopharyngeal and throat swab samples were collected at Siriraj Hospital, Thailand. 

SHERLOCK: Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

Unlike CRISPR, the Cas13a protein needs to be purified and is recombinantly expressed. Target site-recognition activates reporter probes resulting in fluorescence to recognize viral RNA. Assays can be designed as a paper dipstick test which delivers in 30-60 minutes. 


University of California, Santa Cruz

Smith College

- Isothermal Nucleic Acid Amplification
- RPA
- CRISPR
- Cas13a Assay
- Next Generation Sequencing 
- Nanomaterials (nanotrap particles, etc.)
- Wondfo Qualitative Rapid Chromatographic Immunoassay (RCI)

Emerging Methods for Diagnosis of SARS-CoV-2

Dcruz, R. J., Currier, A. W., & Sampson, V. B. (2020). Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). Frontiers in Cell and Developmental Biology, 8. doi:10.3389/fcell.2020.00468

Frontiers in Cell and Developmental Biology

The development of a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity offers great potential for SARS-CoV-2 diagnosis. 


SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Compared to the conventional culture-based or immunoassay-based approaches, a nucleic acid-based approach relies primarily on a strand-displacing polymerase or T7 RNA polymerase at a constant temperature. In particular, the SplintR ligase can efficiently ligate two DNA probes using a target single-stranded RNA as a splint, enabling the sequence-specific detection of RNA molecules. 


SENSR integrates all component reaction steps using the specially designed probes that contain all required functional parts: promoter, hybridization sequence to target, and an aptamer template. Even with the multifaceted features of the SENSR probes, the design process is systematic and straightforward. The probe design is unique in that two DNA probes are designed to expose single-stranded target recognition parts, enabling hybridization of the target RNA and the probes at 37°C. 


SENsitive Splint-based one-step isothermal RNA detection (SENSR)

This method amplifies a DNA or RNA target sequence, but unlike PCR, does not need thermal cycling. These assays use isothermal methods to allow for primer binding and then amplify the selected strands using a polymerase with stand displacement activity. The amplified gene products are then detected via photometry. 

Isothermal Nucleic Acid Amplification

This method detects the total viral RNA from a cell culture supernatant and 19 nasopharyngeal swab samples. Uses isothermal methods for reverse transcription and then uses recombinase activity to promote primer binding to the respective dsDNA sequence. This process also achieves 100% specificity and sensitivity and offers advantages over the typical RT-PCR in efficiency and portability allowing for clinical decisions to be made real time when a patient visits. 

Uses recently discovered crRNA guided detection that is specific to viral RNA. The Cas13a enzyme that recognizes and binds targeted RNAs in a sequence-specific manner for signal amplification and nucleic acid detection.   

CRISPR

Cas13a Assay

NGS can sequence 30,000 nucleotides of the viral genome. This method provides insight on environmental monitoring, surveillance testing, and origin of the strain to pinpoint the viral evolution. 

Next Generation Sequencing

Diagnostic Testing methods that amplify DNA using reverse transcriptase.
Nucleic Acid Amplification Tests include but are not limited to:

- RT-PCR
- RT-LAMP
- Next Generation Sequencing
- Isothermal Nucleic Acid Amplification
- RPA

NAAT

Biosensors for COVID-19

Combining the excellent electrical and optical properties of nanomaterials with biological or synthetic molecules as receptors to detect selectively any kind of analyte, nanobiosensors are a valuable alternative to conventional diagnostic tools. 

- Graphene-based Field-Effect Transistor (FET) Biosensor

- Dual-Functional Plasmonic Biosensor

Paper/nanomaterial-based sensors, in general, and, particularly, NP-based lateral-flow devices that are observed with the naked eye or operated through a smartphone are now the most reported testing platforms for the analysis of COVID-19-related biomarkers. Nanopaper and nanochannels may also offer cost-efficient alternatives for detection of viruses and related biomarkers. 

Nanomaterials for COVID-19 Diagnosis

The Wondfo RCI functions by detecting IgM/IgG antibodies for SARS-CoV-2.  10 μL of plasma or serum are inserted into the testing device, and antibodies that fight against SARS-CoV-2 contained in the sample attach to recombinant antigens that cover colloidal gold particles, ultimately creating a complex of antigen-antibody/colloidal gold particles.  80 μL of buffer solution must also be inserted into the testing device under the sample cavity.  The results of the RCI can be read within 15 minutes.  A major limitation of RCI is that it is not capable of differentiating between acute disease and infection from the past.  


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