Reverse transcriptase-PCR (RT-PCR) is a nucleic acid amplification test (NAAT) designed to detect RNA viruses in patient samples. In this procedure, the enzyme reverse transcriptase (RT) is first used to synthesize complementary DNA (cDNA) from the small amount of viral RNA present in the specimen, and the resulting cDNA is then amplified via polymerase chain reaction (PCR) to confirm the presence of the viral nucleic acid.

RT-PCR

RT-LAMP functions as using multiple loop primers target the ORF1ab gene and the S gene used for DNA strand displacement activity and then target amplification is used. This method has reported a 100% sensitivity and 100% specificity, and the mean detection time was under 30 minutes. Additionally, RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. This permits labs to extend their capacity and process two times more clinical samples. The technique could potentially be used for high-throughput screening purposes. 

RT-LAMP

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

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. 

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

Nucleic acid amplification tests (NAAT) are diagnostic methods in molecular biology used to identify viruses in patient specimens by detecting unique viral nucleic acid sequences. These tests work by amplifying specific segments of viral DNA or RNA, enabling the confirmation and identification of the target virus.

University of California, Santa Cruz

Smith College

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Currently Used Methods for Diagnosis

- 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

Wikipedia

Harahwa, T. A., Yau, T. H., Lim-Cooke, M., Al-Haddi, S., Zeinah, M., & Harky, A. (2020). The optimal diagnostic methods for COVID-19. Diagnosis, 0(0). https://doi.org/10.1515/dx-2020-0058

The optimal diagnostic methods for COVID-19 (Harahwa et al., 2020)

https://openstax.org/details/books/microbiology/

Microbiology covers the scope and sequence requirements for a single-semester microbiology course for non-majors. The book presents the core concepts of microbiology with a focus on applications for careers in allied health. The pedagogical features of the text make the material interesting and accessible while maintaining the career-application focus and scientific rigor inherent in the subject matter. Microbiology’s art program enhances students’ understanding of concepts through clear and effective illustrations, diagrams, and photographs.

Microbiology is produced through a collaborative publishing agreement between OpenStax and the American Society for Microbiology Press. The book aligns with the curriculum guidelines of the American Society for Microbiology.

OpenStax Microbiology Textbook

Detection Methods Used Currently by Manufacturers

(Please indicate "current" date if not stated elsewhere)

- CDC
- WHO

Detection Methods Used Currently by Organizations

Imaging tests cannot be used as the only reliable diagnostic tool to detect infection due to its lack of sensitivity. However, it can be useful in medical settings with limited resources where the virus is prevalent.

- Chest Radiograph
- Ultrasound
- Computed Tomography

Radiographics Test / CT Imaging for COVID-19

The use of antibody-testing with a similar sensitivity and specificity to RT-PCR. However, the virus is detected in a later state (10-20 days into the infection). 
- SVN
- ELISA
- LFIA


Biomarkers

NAAT

No single diagnostic test is entirely sufficient for diagnosing an active viral infection before death. The accuracy of these assays depends on their detection sensitivity, the quantity of virions present in the sample, and the timing of the test relative to viral shedding, which can vary. Consequently, a negative test result does not definitively rule out an infection, as viral loads may simply be too low at the time of sampling, often requiring repeated testing over time to ensure detection.

Limitations of Viral Diagnostic Testing

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)

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

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. 


Cas13a Assay

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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