simple overview of RT-PCR technique for COVID-19 testing

1. Presented by
RIPU Chandra Das
Department of microbiology
Noakhali science and technology university.

2. WHAT IS REAL TIME rt-PCR?
 A real-time reverse transcription polymerase chain reaction is
a laboratory technique of molecular biology based on the polymerase
chain reaction (PCR).
 It monitors visualized the amplification of a targeted nucleic acid
molecule during the PCR (i.e., in real time) not at its end, as in
conventional PCR.
 Reverse transcription(conversion of RNA to DNA) is occur.
 Two common methods for the detection of PCR products in real-time
PCR are
 (1) non-specific fluoroscent dyes that intercalate with any double-
stranded DNA.
 (2) sequence-specific DNA probe consisting of oligonucleotides
that are labelled with a fluoroscent reporter, which permits
detection only after hybridization of the probe with its
complementary sequence.

3. BASIS OF RT-PCR FLUOROSCENT EMISSION

4. Target gene and fluorophore setup
IC (internal control)- for confirming the presence of
human sample-cy5 chromophore.
N gene- viral gene- ROX chromophore.
ORF1ab – viral gene- FAM chromophore.

5. REAL TIME RT-PCR SIMPLE PROCEDURE
Run the thermal cycler and result is interpretated
Fluorophore for target gene is also set
Then PCR plate is being set and temperature is also set
30μL of master mix (PCR mix + enzyme mix)is added
10 minutes room temperature incubation
10μL of sample is added in lysis buffer through gentle pipetting
Collected sample is vortexed for 3-5 seconds
10μL lysis buffer is added in each PCR tube

6. REAGENT FOR PERFORMING RT-PCR

7. REAL TIME RT-PCR CONDITION:

8. RESULT INTERPRETATION
 Our main target is to find the Ct value which is defined
as the number of cycles required for the fluorescent signal
to cross the threshold (i.e. exceeds background level)
According to Sansure Kit, Ct value <40 for N gene and
ORF1 ab should be considered as positive
But as the kit is assigned for qualitative and rapid
identification, there is a chance of false positive result by
taking the border line result (Ct>37)

9. FIGURE OF POSITVE SAMPLE

10. FIGURE OF NEGATIVE SAMPLE

11. THANK YOU