This document describes the SARS-CoV-2 Real-TM test, which is a real-time PCR test for detecting SARS-CoV-2 RNA in clinical samples. The test involves three major processes: isolation of viral RNA from specimens, reverse transcription of the RNA into cDNA, and real-time PCR amplification of the cDNA. It is a multiplex real-time PCR assay that detects SARS-CoV-2 through probes targeting the N and E genes of the virus. Results are analyzed by examining fluorescence of probes for the N and E genes, and the test includes controls for extraction, amplification, and RNA integrity.

1. GKM
Ahmed
SARS-CoV-2 Real-TM

2. SARS-CoV-2 Real-TM
SARS-CoV-2
Coronaviruses are a large family of ribonucleic acid (RNA) viruses.
is a new strain which has not previously been identified in humans.
known also as 2019 novel coronavirus(2019-nCoV).
Severe acute respiratory syndrome coronavirus 2
SARS-CoV-2 Real-TM
is Real-Time PCR test for the Qualitative detection of SARS-CoV-2 (COVID-19
virus, 2019-nCoV) RNA in clinical samples.

3. SARS-CoV-2 Real-TM
Sa Cycler-96

4. SARS-CoV-2 Real-TM
SARS-CoV-2 Real-TM
is based on three major processes
# The reverse transcription and amplification are performed in a single,
one step reaction.
isolation of virus RNA from specimens
reverse transcription of the RNA
Real Time PCR amplification of the cDNA.
RNA cDNA

5. SARS-CoV-2 Real-TM
SARS-CoV-2
E gene
Region E geneSARS-CoV-2
SARS-like coronaviruses
ROX/Orange
Cy5/Red
FAM/Green
IC - Internal Control JOE/HEX/Yellow
SARS-CoV-2 Real-TM.
multiplex Real-time PCR assay
N gene These dyes are linked with
probes of oligonucleotides
which bind specifically to
the amplified product

6. SARS-CoV-2 Real-TM
isolation of virus RNA
DNA/RNA Prep Sacace
DNA/RNA Prep NA Sacace
QIAmp™ DSP Viral RNA Mini Kit Qiagen®
SaMag Viral Nucleic Acids Extraction kit Sacace
SaMag Viral/Bacterial Nucleic Acids Extraction Kit B Sacace
RNA
Extraction
Internal Control RNA(IC)
Lysis mixture

7. SARS-CoV-2 Real-TM
Format T (tube format)
1 Tubes-COVID19, 96 tubes 2 RT-PCR Buffer
3 Enzymes Taq/RT 4 Pos cDNA C+
5 Negative Control 6 Internal Control RNA
Contains reagents for 96 tests
Format S (strip format)
1 Strips-COVID19, 8x12 strip tubes 2 RT-PCR Buffer
3 Enzymes Taq/RT 4 Pos cDNA C+
5 Negative Control 6 Internal Control RNA
Contains reagents for 96 tests

8. SARS-CoV-2 Real-TM
ONE STEP REVERSE TRANSCRIPTION AND PCR AMPLIFICATION
Negative control of extraction (NCE),
Negative control of amplification (C-)
Positive control of amplification (C+)
Reaction Mix
Enzymes Taq/RT 0.5ul *N
RT-PCR Buffer 15ul *N
Vortex
spin briefly for 3-5 sec
15 μl RNA

9. SARS-CoV-2 Real-TMRESULTS ANALYSIS
Positive SARS-Cov-2 RNA ROX/Orange Cy5/RedFAM/Green
HEX/Yellow
Cy5/RedFAM/Green
ROX/OrangeFAM/Green
Negative SARS-Cov-2 RNA
positive SARS-like coronaviruses
RNA
FAM/Green
doubtful
Cy5/Red
ROX/Orange
sample is considered invalid and
the test should be repeated starting
from the RNA extraction stage.
E geneRegion E gene N gene