PCR, RT PCR and Rapid Antigen Testing Flashcards
State the stages of PCR
The stages of PCR are denaturing, annealing, and extending (1)
These three stages help amplify a specific sample of DNA (2)
The denaturing of DNA is possible as a result of high temperatures, breaking apart the hydrogen bonds that form the base pairs (3)
After the hydrogen bonds are broken, primers attach onto the DNA through complementary pairing (4)
Finally, the state of extending is when temperatures are increased (5) and Taq polymerase enzyme allows the primer to extend and add nucleotides to synthesize new strands of DNA (6)
State the reagents required for PCR
- (1) DNA template
- (2) forward and reverse primers
- (3) DNA Taq Polymerase
- (4) Deoxynucleotide triphosphates (dNTPs)
- (5) Reaction Buffer
Explain how PCR generates large quantities of a target DNA sequence
A target DNA sequence is first denatured at 96 degrees Celsius (1)where DNA is separated into a single strand through the weakening of hydrogen bonds (2)
The reaction is cooled between 55 - 65 degrees to allow forward and reverse primers to bind to complementary sequences (3). The primers are each used in PCR reaction to flank the target region (4)
Extension occurs at 72 degrees, allowing Taq polymerase to attach to primers and extend nucleotides (5)
Describe real time reverse transcription PCR (real time RT PCR)
Real time reverse transcription PCR (real time RT PCR) is a live display of DNA that is possible as the guide RNA turns into c-DNA.
Reverse transcriptase (RT) is a DNA polymerase enzyme that transcribes single stranded RNA into DNA and synthesize a double helix DNA once RNA has been reverse transcribed.
The same process is used, except RT-PCR has an added step of reverse transcription of RNA to DNA to allow for the amplification of DNA.
Define an antigen and an antibody
An antigen is a substance that causes the body to respond to it (1). Infectious antigens include bacteria, viruses, protozoal, etc. (2) Antigens may be simple or complex, protein,
carbohydrate, or synthetic in origin (3)
An antibody, also known as immunoglobin are molecules found in blood and other bodily fluids (1) and are used to identify and destroy antigens (2)
Explain how rapid antigen tests work
Liquid containing antigens are collected via swab and placed onto an antigen test (1)
Liquid travels along the strip and if a virus is present in the liquid the antibodies become stuck onto the strip (2)
The results become positive if the antibody complex gets stuck onto the test strip and a reaction occurs (3)
Reaction may include fluorescence or color change
If the liquid is negative, the antibody contains liquid but does not stick to the strip (4)
Evaluate the pros and cons of RT-PCR
The pros of RT-PCR:
- Detects active infections
- Quick to define targets and to design primers
- Can work from simple tissue swabs from nasal or cheek
- POC options available
The cons of RT-PCR
- Cannot detect past infections
- Are best suited to centralized labs
- Take long time to complete
- Accuracy is dependent on viral load
- Specificity (cross - reactivity with other viruses)
