Question

A 30-year old woman presents to the ER in late December with fever, dry cough and tiredness. She reported being in contact with a person having flu-like symptoms

1-    1--  Give two possible viral diagnosis for her case.

2-     2- What is the confirmatory test that should be performed?

3-     3- What are the steps of this confirmatory test? State the components (example reagents) of each step.

4-     4- What is the difference between this confirmatory test and the one used for diagnosing Herpes virus?

Answer 1

1. Two possible viral diagnosis:

1. Coronavirus disease

2. Influenza (flu)

3. Pneumonia

2. What is the confirmatory test that should be performed:

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is the gold standard frontline test for coronavirus (COVID-19)

3. What are the steps of this confirmatory test? State the components (example reagents) of each step.

RT-PCR Process

• After RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.
• If the target is present, primers anneal to the RNA strand.
• Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.
• The temperature is raised to 95^o C, and the RNA/DNA strands are denatured.
• The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.
• Polymerase enzyme synthesizes a new DNA strand, extending from the primer.
• Multiple cycles geometrically increase the number of copies of DNA.

RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40^o C and 50^o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.

4.

The earliest applications of real-time PCR for testing in the clnical microbiology laboratory were reported for the detection of viruses. This was not unexpected as conventional PCR assays were already recognized as the method of choice for detecting or quantifying some viruses, (e.g., detection of herpes simplex virus in cerebrospinal fluid (CSF) or quantification of cytomegalovirus in blood or plasma).

In PCR:

For standard PCR, all that is need is a DNA polymerase, magnesium, nucleotides, primers, the DNA template to be amplified and a thermocycler. The PCR mechanism is as simple as its purpose: 1) double-stranded DNA (dsDNA) is heat denatured, 2) primers align to the single DNA strands and 3) the primers are extended by DNA polymerase, resulting in two copies of the original DNA strand. The denaturation, annealing, and elongation process over a series of temperatures and times is known as one cycle of amplification. Each step of the cycle should be optimized for the template and primer set used. This cycle is repeated approximately 20-40 times and the amplified product can then be analyzed. PCR is widely used to amplify DNA for subsequent experimental use. PCR also has applications in genetic testing or for the detection of pathogenic DNA.

But in RT-PCR:

RT-PCR

Reverse transcription PCR, or RT-PCR, allows the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template is essential for the success of RT-PCR. The first step of RT-PCR is the synthesis of a DNA/RNA hybrid. Reverse transcriptase also has an RNase H function, which degrades the RNA portion of the hybrid. The single stranded DNA molecule is then completed by the DNA-dependent DNA polymerase activity of the reverse transcriptase into cDNA. The efficiency of the first-strand reaction can affect the amplification process. From here on, the standard PCR procedure is used to amplify the cDNA. The possibility to revert RNA into cDNA by RT-PCR has many advantages. RNA is single-stranded and very unstable, which makes it difficult to work with. Most commonly, it serves as a first step in qPCR, which quantifies RNA transcripts in a biological sample.