RT-PCR

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

HOW RT-PCR IS DONE?

• 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. 

Microcentrifuge tube.

• Then it's mixed with a lysis buffer. This buffer is highly denaturing and is usually consists of phenol, and guanidine isothiocyanate.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.

Pulse vortexing.

• 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.
• d 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.
• 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.
• 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.