"Comprehensive RT-PCR Analysis for the Detection and Quantification of Viral RNA in Diagnostic Testing"

Real-Time PCR, also known as quantitative PCR (qPCR), is a molecular biology technique used to amplify and simultaneously quantify a targeted DNA molecule. Unlike traditional PCR, which only allows for the detection of DNA after amplification, Real-Time PCR enables the monitoring of the amplification process in real time. This is achieved through the use of fluorescent dyes or probes that emit signals proportional to the amount of DNA produced during each cycle of the PCR process.

Real-Time PCR is widely utilized in various applications, including gene expression analysis, pathogen detection, genetic variation studies, and quantification of nucleic acids in research and clinical laboratories. Its high sensitivity, specificity, and ability to provide quantitative data make it an essential tool for both basic research and clinical diagnostics, allowing for precise measurement of DNA or RNA levels in complex biological samples.

1. History Principle Extraction Master Mix Procedure Result Application AGENDA

2. * Image of American Biochemist Kary Mullis. History The invention of PCR in 1983is generally credited to‘Kery Mullis. He was awarded the Nobel Prize in chemistry in 1993 for this invention. The discovery in 1976 of Taq - Polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus , which naturally lives in hot condition (50-80 °C)

3. The Principal of PCR : PCR employs  DNA polymerase  to add a nucleotide to the 3' end of a pre-existing short DNA fragment called a  primer  to generate new DNA complementary to the template strand. Basic steps in PCR include: Denaturation Annealing Extension These steps are performed at different temperatures and repeated in multiple cycles, hence the name  "chain reaction." Principle of PCR

4. SAMPLE EXTRACTION Vortex all sample property. 2.Take 10 μL Lysis buffer each PCR Reaction Tube. 3.Mix sample with lysis bufffer by pipetting. 4.Incubation for 10 minutes at Room Temperature. (Inside the Biosafety Cabinet) Transfer to Master Mix Room CLASS II BIOSAFETY CABINET

5. Master Mix PCR Mix Contain are: Primer DNTP ( Deoxynucleotide triphosphate) MgCl ₂ Taq polymerase enzyme PCR Enzyme Preparation of Master Mix : * PCR Mix 26μL and 4 μL PCR Enzyme

6. Master Mix Positive Control Positive Control 20 μL + Master Mix 30 μL = Total 50 μL Negative Control Negative Control 20 μL + Master Mix 30 μL = Total 50 μL Sample Sample 10 μL + Lysis Baffer 10 μL + Master Mix 30 μL = Total 50 μL Ready to Run Sample

7. Control: Positive Control : Negative Control

8. Result : Positive Result : Negative Result

9. COVID -19 virus detection by RT-PCR Hepatitis B Virus (HBV) DNA detection Hepatitis C Virus (HCV) RNA Test (Quantitative) Hepatitis C Virus RNA Genotyping Human Papillomavirus (HPV) detection Human leukocyte antigen B27 (HLA-B27) detection Cytomegalovirus (CMV) DNA detection Mycobacterium tuberculosis (MTB)-PCR (Qualitative) etc. Common RT - PCR in Our Country