A short introduction to reverse transcription polymerase chain reaction

1. A SEMINAR ON
REVERSE TRANSCRIPTASE POLYMERASE
CHAIN REACTION (RT-PCR)TESTING
METHOD FOR COVID-19
PRESENTATION BY: ADONGOI, David Llyod
DEPARTMENT OF BIOCHEMISTRY
FOS/19/20/258725
DELTA STATE UNIVERSITY ABRAKA, DELTA
STATE.
FEBRUARY, 2023.

2. OUTLINE
Introduction
Requirement
Molecular targets
COVID-19 Detection
Interpretation of molecular
results
Conclusion

3. Introduction
Coronavirus Disease (COVID-19) is an illness
caused by a new strain of coronavirus called
Severe Acute Respiratory Syndrome Coronavirus
2 (SARS-CoV-2) that has been identified in
humans.
The virus is a positive – sense single – stranded
RNA virus.

4. Contd
The real-time reverse transcription-
polymerase chain reaction (RT-PCR) is the
molecular-based assay used globally to
detect SARS-CoV-2 RNA in clinical samples
of patients manifesting COVID-19
compatible signs and symptoms.

5. Contd
• This technique can be performed in ‘real time’
• The method uses fluorescent dyes to detect
targeted genetic materials (a specific part of the
transcribed viral DNA)

6. Requirements

7. Molecular Targets
A set of primers and probes has been designed for three
regions; RdRP gene, E gene and N gene
At least two molecular targets are used to avoid
potential cross-reaction with other endemic
coronaviruses as well as potential genetic drift of
SARS-CoV-2

8. COVID-19 Detection
• The basic idea behind fluorescence-based real-time
RT-PCR assay is straight-forward. After RNA is
reverse transcribed into cDNA; a suitable detection
chemistry (Taqman Probe) reports the presence of
PCR products; an instrument monitors the
amplification in real time; and an appropriate
software analyzes the data.

10. Interpretation of Molecular
results
In this technique, viral RNA is measured by cycle
threshold (Ct), which is the number of cycles of
amplification required to generate a fluorescent signal that
exceeds a baseline level
A Ct value less than 37 is defined as a positive test while
a Ct value of 40 or more is defined as a negative test

12. Conclusion
• The real time RT-PCR remains the most sensitive
technique for confirming COVID-19 disease in all
affected countries. Therefore, adequate knowledge of real
time RT-PCR is critical for accurate implementation of the
assay.
• Finally, the results of real time RT-PCR tests must be
cautiously interpreted

13. THANKS FOR
LISTENING