When a person is infected, the most common symptoms include fever, cough, and shortness of breath

To start a test

The samples can be collected by a nasopharyngeal swab or an oropharyngeal swab. For Nasopharyngeal specimen

the swab is inserted in the nostril and gently moved forward into the nasopharynx

then it is rotated for a specified period time to collect secretions that contain the virus

Once the swabbing is applied, the swab is placed immediately into sterile tube containing a viral transport medium

The standard method of coronavirus testing is polymerase chain reaction, PCR

Which is a method that used widely in molecular biology to make millions to billions of copies of a specific DNA fragment rapidly

Coronaviruses contain an extraordinarily long single-stranded RNA genome

To detect these viruses with PCR, RNA molecules must be converted into their complementary DNA sequences by reverse transcriptase

Then the newly synthesized DNA can be amplified by standard PCR procedures

This approach is universally known as RT-PCR

To perform this method, basically viral RNA should be extracted

Several RNA purification kits are available for convenient, fast and effective isolation

To extract the viral RNA by using commercial kit

the sample is first added into a microcentrifuge tube. Then it's mixed with a lysis buffer

This buffer is highly denaturing and is usually consists of phenol, and guanidine isothiocyanate

Also, RNase inhibitors are usually present in the lysis buffer to ensure isolation of intact viral RNA

Once the lysis buffer is added, the tube is mixed by pulse-vortexing, and incubated at room temperature

Then the virus is lysed under the highly denaturing conditions provided by the lysis buffer

Once the sample is lysed, a purification procedure is carried out by using a Spin column

the sample is loaded onto the spin column

then a centrifugation is performed

This procedure is a solid phase extraction method, in which the stationary phase consists of a silica matrix

Under optimal salt and pH conditions, RNA molecules are bind to the silica gel membrane, and at the same time

protein and other contaminants are not retained

After centrifugation, the spin column is placed into a clean collection tube, and the filtrate is discarded. Then a wash buffer is added

The column is put in a centrifuge again, forcing the wash buffer through the membrane.This removes any remaining impurities from the membrane

leaving only the RNA bound to the silica gel

Once the sample is washed, the column is placed in a clean microcentrifuge tube, and an elution buffer is added

Then a centrifugation is carried out, forcing the elution buffer through the membrane

The elution buffer removes the viral RNA from the spin column

And a purified RNA, which is free of protein, inhibitors, and other contaminants is obtained

After the extraction of the viral RNA, the next step is the preparation of the reaction mixture for PCR amplification

In this step, a master mix is used which is a premixed concentrated solution, that consists of buffer, Reverse Transcriptase enzyme

Nucleotides, Forward Primer, Reverse Primer, TaqMan probe, and DNA polymerase

Finally, to complete this reaction mixture, the RNA template is added

The tube is Mixed by pulse-vortexing

then the reaction mixture is loaded into a PCR plate, which generally contain 96 wells

Allowing the analysis of several samples at the same time

Next, the plate is placed in a PCR machine, which is essentially a thermal cycler

Real-time RT-PCR is used for the detection of the new coronavirus 2019

by the amplification of target sequences in the Rdrp gene, the E gene and the N gene

The choice of the target gene depends on the primers and the probe sequences

The first step in RT-PCR is reverse transcription

The first-strand complementary DNA synthesis, is primed with the PCR reverse primer

which hybridizes to a complementary part of the virus RNA genome

Reverse transcriptase then adds DNA nucleotides onto the 3-prime end of the primer

Synthesizing DNA complementary of the viral RNA

The temperature and duration of this step depend on the primer, the target RNA and the reverse transcriptase used

Next, an initial denaturation step is applied, causing denaturation of the RNA-DNA hybrids

This step is required for the activation of DNA polymerase

and simultaneously the inactivation of reverse transcriptase

PCR consists of a series of thermal cycles, with each cycle consisting of Denaturation, Annealing, and Extension steps

Denaturation step consists of heating the reaction chamber to 95 degree Celsius.

And it is used for denaturation of the double-stranded DNA template

In the next step

the reaction temperature is lowered to 58 degree Celsius

allowing annealing of the forward primer to its complementary part of the single-stranded DNA template

The annealing temperature relies directly on length and composition of the primers

In the extension step, the DNA polymerase synthesizes a new DNA strand

complementary to the DNA template strand

by adding free Nucleotides from the reaction mixture that are complementary to the template in the 5' to 3' direction

The temperature at this step depends on the DNA polymerase used

After the first cycle, the double-stranded DNA target is obtained

Then, the denaturation of this double-stranded DNA is performed

yielding two single-stranded DNA molecules

In the next step, the reaction temperature is lowered, allowing annealing of the primers to each of the single-stranded DNA templates

and annealing of the Taq-man probe to its complementary part of the target DNA

TaqMan probe consists of a fluorophore covalently attached to the 5' end of the oligonucleotide probe

the fluorescence is emitted by the fluorophore when is excited by the cycler's light source

Also, this probe consists of a quencher at the 3' end

The close proximity of the reporter to the quencher prevents detection of its fluorescence

In the extension step, DNA polymerase synthesizes new strands. When the polymerase reaches a TaqMan probe

its endogenous 5' nuclease activity cleaves the probe, separating the dye from the quencher

With each cycle of PCR, more dye molecules are released

resulting in an increase in fluorescence intensity proportional to the amount of amplicon synthesized

This method allows the estimation of the amount of a given sequence present in a sample

The number of double stranded DNA pieces is doubled in each cycle

therefore, PCR can be used to analyze extremely small amounts of sample.

For the measurement of the fluorescence signal

a Tungsten- Halogen lamp

an Excitation filter, Mirrors, lens, an Emission filter

and a Charge-coupled device - CCD camera are used

Filtered light from the lamp is reflected off mirror, passes through a condensing lens, and is focused into the center of each well

then Fluorescent light emitted from the wells reflects off the mirror, passes through an emission filter

and is detected by the CCD camera

In each PCR cycle, Light from excited fluorophore can be detected by the CCD

which converts the light that it captures into digital data

This method is known as real time PCR

which allows the monitoring of the progress of the PCR reaction as it occurs in real time