1. What is the basic principle of RT-PCR ? 2. What are the components of the master mix you prepare for RT PCR ? 3. What is donning and doffing? 4. What is the biosafety level you require to handle infectious virus like SARS-CoV-2? 5. What is the controls you would choose to validate your PCR/RT-PCR result?

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1. Basic Principle of RT-PCR (Reverse Transcription Polymerase Chain Reaction): RT-PCR combines reverse transcription and PCR. It begins with the conversion of RNA into complementary DNA (cDNA) using reverse transcriptase. The cDNA is then amplified through PCR to quantify the RNA present. This method is commonly used to detect and quantify RNA viruses, such as SARS-CoV-2.

2. Components of the Master Mix for RT-PCR:

Reverse Transcriptase enzyme: Converts RNA to cDNA.

RNA Template: The RNA extracted from the sample.

Primers: Short DNA sequences that bind to specific regions of the cDNA to initiate amplification.

dNTPs (Deoxynucleotide Triphosphates): Building blocks for DNA synthesis.

Buffer solution: Maintains optimal pH and ionic conditions.

MgCl2: Cofactor for the reverse transcriptase and DNA polymerase.

Taq Polymerase (for PCR phase): Enzyme that synthesizes DNA strands.

RNase Inhibitors: Prevent degradation of RNA.

3. Donning and Doffing:

Donning refers to the process of putting on personal protective equipment (PPE), such as gloves, gowns, masks, and face shields.

Doffing is the process of safely removing PPE to prevent contamination and exposure to infectious agents.

4. Biosafety Level for Handling Infectious Viruses like SARS-CoV-2: Handling infectious viruses like SARS-CoV-2 requires Biosafety Level 3 (BSL-3). This level includes strict access control, the use of biosafety cabinets, PPE, and protocols to contain aerosolized pathogens.

5. Controls to Validate PCR/RT-PCR Results:

Positive control: Ensures the reaction worked properly by including a known sample that contains the target RNA.

Negative control (No Template Control, NTC): Confirms the absence of contamination by including all reagents except the template RNA.

Internal control: Ensures RNA extraction and RT-PCR are working by using a housekeeping gene or other internal RNA targets.

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