Real-time RT-PCR in COVID-19 detection: issues affecting the results. (2020, August 21). PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189409/

Real-time RT-PCR in COVID-19 detection: issues affecting the results

The accuracy of COVID-19 RT-qPCR testing is influenced by several key variables:
- **Viral Mutations**: SARS-CoV-2 is known for rapid evolution and genetic drift. Mutations in primer target sequences can cause mismatches, leading to false negatives or positives.
- **Sample Source**: Nasal and throat swabs are most commonly used for patient comfort. Bronchoalveolar lavage fluid (BALF) offers higher accuracy but requires a medical suction device and a licensed operator. Non-respiratory samples like stool and blood can also be utilized.
- **Timing and Handling**: The phase of infection during collection and prompt, careful transport to testing sites are critical to prevent false reads.

Factors Affecting COVID-19 RT-qPCR Accuracy

Reverse transcriptase-coupled quantitative PCR (RT-qPCR) is a rapid and stable nucleic acid amplification test used for early COVID-19 detection. It utilizes viral-specific forward and reverse primers targeting conserved SARS-CoV-2 sequences to achieve high sensitivity and specificity. Despite its overall reliability, the accuracy of RT-qPCR can be compromised by several variables, including viral genetic mutations, sample sources, and the timing of sample collection.

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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

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.

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