False positives can be caused from the cross-reactivity of primers with nucleic acids which happen because contamination of other bacteria and viruses. If reagents of RT-PCR false positives can also occur which is a problem considering the high volume of testing occurirng in laboratories during the pandemic. 
A simpler method of diagnosing COVID-19 where resources are limited would be more beneficial.

Disadvantages of RT-PCR

Advantages: RT-PCR can analyze thousands of samples in a day at a sensitivity of 95%. Positives should generally follow the trend of clinical observations and epidemiological information.

Advantages of RT-PCR

RT-PCR can be performed in either a one-step or a two-step
assay. 

In a one-step assay, reverse transcription and PCR
amplification are consolidated into one reaction. This assay format can provide rapid and reproducible results for high-throughput analysis. The challenge is the difficulty in optimizing the reverse transcription and amplification steps
as they occur simultaneously, which leads to lower target
amplicon generation. 

In the two-step assay, the reaction is done sequentially in separate tubes. This assay format is more sensitive than the one-step assay, but it is more time consuming and requires optimizing additional parameters.

Controls need to be carefully selected to ensure the reliability of the assay and to identify experimental errors.

One-step and Two-step RT-PCR Assays

RNA is reverse transcribed into cDNA and amplified using a polymerase chain reaction. Primers target various sections of the virus genetic sequence with the E gene being the highest sensitivity test and being confirmed by the RdRp gene.
Upper and lower respiratory samples are collected. Nasopharyngeal swabs are high priority specimens for SARS-CoV-2 while low priority specimens include: sputum, oropharyngeal swabs, and tracheal aspirates.

University of California, Santa Cruz

Smith College

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

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

Udugama B, Kadhiresan P, Kozlowski H, Malekjahani A, Osborne M, Li V, Chen H, Mubareka S, Gubbay J, and Chan W

doi: 10.1021/acsnano.0c02624

Diagnosing COVID-19: The Disease and Tools for Detection

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. 

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