RT PCR and RDT

                                                

What is Reverse Transcription-Polymerase Chain Reaction (RT-PCR) or Real time-PCR?

Every living cell is composed of genetic materials either in the form of RNA or DNA which are transferred to the progeny.

DNA is a double stranded macro-molecular structure found in all living organisms (plants, animals and viruses). It holds genetic code which are the blue prints for the transmission of characters from parents to the offspring. It contains four types of nitrogen bases namely Adenine(A), Thymine(T), Cytosine(C) and Guanine(G).

RNA is generally a single strand macromolecule molecule that copies transcribe and transmits parts of the genetic code to proteins so they can synthesize and carry out functions that keep organisms alive and developing. In the virus it acts as genetic material. It is composed of nitrogen bases namely Adenine(A), Uracil(U), Cytosine(C) and Guanine(G).

Viruses like Coronavirus (SARS-Cov2/Covid-19) contains RNA as a genetic material. They intervene healthy cells and multiply their number inside the cells. It takes control over the cell and reprogramme the cell by using transcriptase enzyme from the cell and make the cells- virus producing factories.

In order to decect the early stage of coronavirus infection in the body RT-PCR is an effective method. In this method RNA of the virus is converted into DNA by the process called Reverse transcription method. Actually DNA transcript RNA but in this case RNA is transcript into DNA. So, it is called reverse transcription method. This method is applicable since DNA can be amplified in large amount to detect viruses.  In this method hundreds of thousands of copies of transcribed viral DNA is made.  Amplification of DNA is necessary since it is easier to confirm the viral infection by using a large number of copies than a tiny part of DNA.

How does real-time RT-PCR work with the coronavirus?

 A sample is collected from a person’s nasal or throat swab. The sample is treated with several chemical solutions that remove substances, such as proteins and fats present in the virus which make the outer envelope of the virus, and extracts only the RNA present in the sample.

Prior to the reverse transcription, the extracted RNA is heated to 70⁰C in order to obtain a liner RNA from a coiled one. Then a primer is used in it and it is immediately chilled so that primer gets fixed with the linear RNA. A primer is a short single stranded nucleic acid which initiate the DNA Synthesis.

The RNA is reverse transcribed to DNA using a specific enzyme name reverse transcriptase which generates a single stranded complementary DNA (cDNA) from the viral RNA. Then the denaturation is done by heating upto 95⁰C to separate DNA and RNA. The single strand Complementary DNA is taken as a template for the production of double stranded cDNA by using a single stretch of DNA known as primer.  The cycle repeats over and over to continue copying the target sections of viral DNA. Each cycle doubles the previous amount: two copies become four, four copies become eight, and so on. A standard real time RT-PCR setup usually goes through 35 cycles, which means that by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample.

Whether the new copies of viral DNA are produced or not, can be detected by using a florescent dye (probe) which gives color. The probe binds with the DNA and emit florescence which can be detected and measured by machine's computer and present in real time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. If there is an increase in the florescence in the machine, it shows the positive result. It means the target region of the viral RNA or DNA (since DNA is generated from RNA) is amplified. It means the person is infected by virus. If there is no change in the fluorescence after 1.5-2.0 hours of running PCR, then it shows negative result and hence the person is not infected by virus. RT-PCR is a sophisticated, highly expensive and time consuming technique but it is more effective test to ensure the infection at early stage.  

Rapid Diagnostic Test (RDT)

It is widely used test which does not require sophisticated technology. It is based on the presence or absence of antibody presence in the blood sample to fight against virus.

It simply consist of a test kit similar to that of pregnancy test kit. Rapid diagnosis test is fast, easier but not as effective or confirmatory test as RT-PCR. It gives arbitrary value. It does not give the result whether a person is currently infected or infected earlier. In this test a RDT test kit is taken, which consist of several regions: control(C), immunoglobulin G (IgG), immunoglobulin M (IgM), Sample(S), Buffer solution(B).

Our body has an immune system which defends against any invaders such as bacteria, virus or fungus. These invaders are called pathogens or antigens. In order to fight against such antigens our body produces antibodies which try to keep away the pathogens. A particular antibody fight against a specific antigen. Thus antibodies are antigen specific. The antibody produced against corona virus is a specific one which is produced when a person is infected by SARS-Cov1 or SARS-Cov2. Thus this test kit does not distinguish between SARS-Cov1 or SARS-Cov2. IgM is a primary response antibody which is produced when a person is infected for the first time. IgG is the antibody which is produced when a person is infected by the virus or bacteria for the second time.

How does RDT works?

In this method simply a sample of blood is taken by pricking a finger tip with a needle and about 10 micro-liter of blood is put in the S region of the kit. A drop of buffer solution is put in region B. The buffer solution is simply a liquid which carry the blood to the upper part of the kit through capillary action. The sample is kept untouched until a line appears in the control section (C). It confirms that the blood has reached upto the top level. If a person has virus in his/her blood, a line is shown in IgM or IgG section. It means that the person is infected by the coronavirus. If only a single line appear in the control section but nothing appears in the IgM or IgG section, then the person is said to be not infected by the virus.