general knowledge about covid-19, RT-PCR, and a short description how to perform RT-PCR test, hope it will be helpful to you.

1. Early signs and Detection of
Corona viruses – RT PCR
By
ARNAB KUMAR SAMANTA

2. OVERVIEW :
Corona viruses are a group of RNA viruses that causes diseases
in mammals and birds. In human this virus causes respiratory
tract infection.
Structure: These are the large , mostly spherical particles
with bulbous surface projection .The average diameter of the
virus particles is around 125nm . The diameter of the envelope
of the virus is 85nm and the spikes are 20nm long .The viral
envelope consists of a lipid bi layer , in which the membrane ,
envelope , and spike structure proteins are anchored .The
corona virus surface spike proteins are homotrimers of the S
protein , which is composed of S1 and S2 subunit. The
homotrimers S proteins is a class 1 fusion protein which
mediates the receptor binding and membrane fusion between
virus and host cell .
Genome of the virus:
Corona virus contain a positive-sense, single stranded
RNA genome. The genome size for corona viruses range
from 26.4to 31.7kilobases. The genome size is one of
the largest among RNA viruses. The genome has a
5’methylated cap and a 3’ polyadenylated tail.
The genome organization for a corona virus is 5’leader-
UTR-replicase/transcriptase-spike –envelope-
membrane-nucleocapsid-3’UTR-poly(A)tail.

3. Symptoms of COVID-19
COVID-19 may not initially causes any symptoms for some people . You may carry the virus for 2 day to up to 2 week
before you notice symptoms.
Some common symptoms that
have been specifically linked to
COVID-19 include:
Fever- 83-99%
Loss of Appetite- 40-84%
Fatigue-44-70%
Loss of smell-15 to 30%
Shortness of breath-31-40%
Cough-59-82%
Coughing up sputum-28-33%
Muscle aches and pain-11-35%
Symptoms for severe condition:
Difficulty waking confusion
Bluish face or lips
Coughing up blood
Persistent chest pain
Decreased white blood cell
Kidney failure
High fever
NOTE:
Most people (about 80%)recover from the disease
without needing hospital treatment .Around 1 out
of every 5 people who gets COVID-19 becomes
seriously ill and develops difficulty breathing
.Older people ,and those with underlying medical
problems like blood pressure, heart and lung
problems diabetes ,or cancer are at higher risk of
developing serious illness .

4. COVID-19 testing methods:
COVID-19 testing can identify the SARS-CoV-2 virus and itself (RT-PCR, isothermal nucleic acid amplification
,antigen)and antibodies that produced in response to infection.
Reverse transcription Polymerase chain
reaction (RT-PCR): This technique is performed by
extracting the viral RNA from sample . In this technique Reverse
transcription is used to convert the extracted RNA into DNA and then
use PCR to amplify the piece of the resulting RNA.
Isothermal amplification assays:
Just like PCR it is also used to amplifying the virus’s genome but at a
faster rate.
Antigen test:
This test is used to identify the surface spikes of corona virus . It is
performed by using the nasal swab to collect the sample from the
nasal cavity.
Serology test:
This test can detect antibody (IgM,IgG) produced in response to
infection .According to the FAD, IgM antibody to SARS-CoV-2 are
generally detectable in blood several days after initial infection . And
IgG antibody to SARS-CoV-2 generally become detectable 10-14 days
after infection . This test is performed by drawn the blood from the
patient.
Medical imaging (chest CT
scan):
This test is used to identify the abnormalities in
lungs .It is not recommended for routine screening .

5. RT-PCR (Reverse transcription polymerase chain reaction):
It is a laboratory technique is a combination of reverse transcription of RNA into DNA (cDNA)and amplification of
specific DNA target using polymerase chain reaction.
Real time RT-PCR:
This is primarily used to measure the amount of a
specific RNA , by monitoring the amplification reaction
using fluorescence in real time
currently there are four different fluorescent DNA
probes available for the real time RT-PCR detection –
SYBR Green , Taqman , molecular beacons and
scorpion probes .
End –point RT-PCR:
End –point RT-PCR is the analysis after all the cycle of PCR are completed .
The use of end –point RT-PCR is preferred for measuring gene expression changes in small number of
sample .
In this process ethidium , bromide , P32 are used as fluorescent dyes
It is achieved using 3 different methods:
Relative , competitive , and comparative.
One step and two step RT-PCR:
RT-PCR technique can be one step or two step reaction .
The difference between these 2 reaction is that in two
step reaction reverse transcriptase reaction and PCR
amplification be performed in separate tubes .On the
other hand the entire reaction from cDNA synthesis to
PCR amplification occurs in a single tube in one step
process.

6. RT –PCR test for COVID-19:
RT-PCR is a nuclear derived method for detecting the pathogen including the virus .Originally this method use
radioactive isotope marker to detect targeted genetic material. This method include following steps-
Specimen collection :
Throat/oro-pharyngeal swab collection:
1. Gently tilt the patient’s head back
2. Insert a sterile swab
3. Swab both tonsils and the posterior pharynx vigorously with a rotating
motion
4. Remove the swab without touching the tongue.
5. Then swab is placed in the labeled tube containing VTM
Nasal swab collection:
1. Take a fresh sterile swab.
2. Insert the swab into the nostril parallel (1-2cm) to the palate until the
resistance is met at turbinate.
3. Hold the swab in that position for few seconds and then withdraw
slowly in a firmly rotating motion.
Sample handling at collection site:
1. A unique specimen ID is written on each VTM sample
2. The sample kept in a cool box immediately after collection
3. Repeated freezing and thawing must be avoided.
Transportation of the sample from field to the
testing laboratory :
1. Sample should be transported at2-8 degree C within specific time
frame to the laboratory.
2. Samples should kept in proper standing position in appropriate rack
3. The VTM containing part of the tube in direct contact with frozen gel
pack.

7. Current testing modalities in laboratory for COVID-19 diagnosis :
Extraction of the viral RNA: there are lots of kits that are used to extract the viral RNA( e.g. Direct- zol RNA Mini
prep kit). The extracted RNA can be used immediately or stored at<-70 degree C.
Process of RT-PCR: The one step RT-PCR is used in this process. The viral RNA is reversed transcribed to cDNA
by a reverse transcriptase enzyme , reverse transcriptase PCR buffer, and by adding primers and probe specific to 2019-
nCoV(e.g. 2019-nCoV_N1 Forward primer-5’-GAC CCC AAA ATC AGC GAA AT-3’ , Reverse primer-5’-TCT GGT TAC TGC
CAG TTG AAT CTG-3’ and 2019-nCoV_N1 probe- 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’). These fragments
attach themselves to the target section of viral DNA if the virus present in the sample . Some of the added genetic
fragments( primers) are for binding DNA strand during amplification , while others(probe) are for identifying the viral
DNA.
The mixture is then placed in RT-PCR machine .The machine
cycles through temperature that heat and cool the mixture to
trigger the specific chemical reaction that create new identical
copies of target section of viral DNA .the cycle repeats over and
over and double the previous amount of viral DNA .A normal real
time RT-PCR setup normally goes through 35 cycles.
As the new copies of viral DNA are formed the
marker levels(probe) attach to the viral DNA and release
fluorescence , which is measured by the machines computer and
presented in real time on the screen . Computer track the
amount of fluorescence per cycle .When the amount goes over a
certain level of fluorescence , this conform the virus is present in
the sample .
Initial screening RT-PCR involves detection of E gene (coding for SARS-CoV2 viral envelop)
Conformation of sample s positive in screening PCR involves detection of one of the following 2 gene target –
•RdRp gene(coding for SARS-CoV-2 RNA dependent RNA polymerase)
•ORF gene (coding for SARS-CoV-2 Open Reading Frame)
NOTE:
Taqman probe are labeled at the 5’end with the reporter
molecule 6-carboxyfluorescein (FAN)and with quencher,
Black Hole Quencher 1 at the 3’-end.

8. Molecular mechanism of RT-PCR :
Viral RNA(extracted
from the sample)
2019 SARS-
CoV-2
specific
primer
70 degree
followed by
quick chilling
Binding of the
primer with the
specific part of
viral RNA
Reverse
Transcriptase
,dNTPs, Buffers
Reverse
Transcriptase bind
with the primer
and synthesizes
the cDNA by
adding dNTPs
cDNA synthesis is
completed
cDN
A 50-
60degree
C
72degr
eeC
Taq
polymerase
2019-SARS-
CoV2
specific
probe
Binding of the probe
with DNA strand
Cycle is completed
and marker release
fluorescence
Showed on the machine’s
computer in real time
Annealing of
DNA
Polymerization

9. Advantages of Real time RT-PCR:
More sensitivity and specificity:
The real time RT-PCR method is more sensitive , specific and efficient . Though the probes and primers
are highly sequence-specific, if any non- specific bindings occurred, it is monitored immediately during
the reaction . Also , the main reaction or the quantification of target template cannot be influenced by
the non-specific binding.
Fewer templates required:
The overall assay required less amount of the template material . It required 1000 folds less template
DNA or RNA for the reaction to occur as compared with the conventional PCR.
It is time-efficient :
Real time-RT-PCR gives results in ultra fast time .The average duration of each real time RT-PCR test is
4 hours.
The method can do quantitative as well as qualitative analysis.
Disadvantages of real time RT-PCR:
The method is extremely sensitive , even a small
amount of contamination can lead to false result .
The RT-PCR technology is fairly expensive method
. The cost of the chemicals and importing elements
required for this test is high . One test can cost a
minimum of Rs4500.