SWU PHINMA

Dorothy Nacilla
PCR Testing in
SARS-CoV2
 Introduction to PCR

Polymerase
Chain Reaction
Testing the presence of viral RNA using real time Reverse
Transcriptase Polymerase Chain Reaction or RT-PCR.

SARS - COV2
real time
RT-PCR
 PCR is a test
to
determine
genetic
material
from specific
organism
such as virus.
 Specimen for SARS-CoV2:
Oropharyngeal and
Nasopharyngeal
Others: Saliva

Extract all RNA in the

sample but RNA is more
difficult to work with in
the lab than DNA.
Overview of real time RT-PCR
DNA can be easily amplified & quantified using simple,
accurate method called PCR or polymerase chain reaction.
PCR does not work with RNA.So,we convert RNA to DNA
using an enzyme called Reverse transcriptase.

Resulting DNA: Complementary DNA ( cDNA )

Sequence is complementary to that of the original RNA
strand.
Note: created a collection of cDNA's representative of all
the RNA present in the original sample

RNA : includes virus,bacteria,patient's own cell

A way to determine presence of SARS-COV2 cDNA : PCR
Invented: 1985 by Kary Mullis in which a small amount of DNA can be
copied in large quantities over a short period of time.

Amplify + detect specific DNA molecule = viral cDNA

3 STEPS: 40x
1. Denaturation : 2 strands separated
2. Annealing: a pair of primers* specific to SARS-CoV2 cDNA
attaches/anneals to each DNA strands
3. Elongation: enzyme polymerase adds nucleotides to the ends of the
primers using the original DNA strand as a template
*Primers are short nucleotide sequences complementary to unique
sequence in the viral cDNA
Specificity of primers ensures that they only bind to viral
cDNA and not to other cDNA present in the sample.

1 double stranded DNA to 2

These 3 steps are repeated with the amount of DNA doubling in every cycle.
In real time pcr, a probe
is added during the pcr
process which gives off
fluorescence whenever
a new DNA molecule is
formed.
 Increase in viral cDNA = increase flourescence signal
When the level of fluorescence exceeds a certain
threshold,the signal is significantly above the background
level.
CT= cycle threshold, is the number of cycles required for
the fluorescent signal to exceed that specific threshold
value.

Inversely proportional: The lower the CT value,the more

RNA present in the original sample indicating higher viral
load.
For SARS-CoV2
 RESULTS +
CLINICAL
CORRELATION
Thank You!