Demystifying PCR Testing

Introduction

‘DNA’ or deoxyribonucleic acid is a term that a few decades ago was jargon used by biologists. But today, it has become commonplace, a part of regular conversation. It is the genetic material that is present in each cell of our body and gets passed on across generations (when people talk about genes and heredity, they are essentially talking about DNA). It is therefore implied that it has a very important function. It has the responsibility of passing on the right information across generations and within the cell, much like a manager in a company. Like a manager, it cannot do all the work itself, so it delegates. This is where the ‘central dogma’ comes into play.

The central dogma of biology explains the flow of information within a cell. It is a two-step process; the first is the passage of information from DNA to RNA or ribonucleic acid. This is called ‘transcription’. The second step is the passage of information from RNA to proteins, a process called ‘translation’. The proteins (and some RNA) are the major workforce in a cell. Each of the two steps requires the assistance of some proteins called enzymes which act as a catalyst for the reactions. To summarise the content so far, the DNA is the genetic material which is the primary information carrier, and it directs RNA, followed by proteins to perform specific functions in the cell. The central dogma applies to almost all organisms, from tiny bacteria to the giant blue whale. But, when we consider viruses, the story is slightly different.

Some viruses, including SARS CoV-2, the infectious agent associated with COVID-19, don’t possess DNA. Instead, RNA acts as the genetic material or primary information carrier in them. What makes the genetic material of each organism unique is its sequence. We can consider DNA or RNA as a beaded string made of four different beads (A, T, G and C; it is noteworthy that RNA possesses U instead of T which is one of the major differences between DNA and RNA). A, T, G and C refer to Adenine, Thymine, Guanine and Cytosine. U refers to Uracil in RNA. The order in which the beads are placed defines the sequence. A unique sequence of DNA or RNA that possesses information for a particular protein is defined as ‘gene’. The most commonly used COVID-19 diagnostic test is based on the detection of the viral RNA or viral genes in the patient samples

The diagnostic test

The first step in the nucleic acid based diagnostic test is the isolation or purification of RNA from the patient samples. SARS CoV-2 infects the upper respiratory tract and hence the sample is collected using throat swabs, which are dipped into a solution. The throat cells of the patient which are attached to the swab get dislodged into the solution, RNA is then purified from this solution.

Once the RNA from the patient sample is purified, the next step is obtaining a corresponding DNA sequence from the RNA, called cDNA (complementary DNA). As mentioned earlier, when information is passed from a DNA sequence to an RNA sequence, the process is called transcription. The reverse is intuitively called Reverse Transcription (RT). This procedure is facilitated by an enzyme/catalyst called reverse transcriptase. Once cDNA is obtained, it is amplified using PCR. Altogether, the procedure is called RT-PCR (Figure 1).

Figure 1: Overview of the RT-PCR Procedure

The amount of viral RNA obtained is much lower than the detection limit. To allow it to reach a detectable level, it needs to be amplified. The method to amplify the amount of a particular DNA sequence is called the polymerase chain reaction (PCR). The ‘polymerase’ here refers to the enzyme or catalyst that brings about the replication or duplication of DNA. In PCR, the replication is done multiple times (exponentially), rendering a lot of DNA at the end (let’s say we start from 2 copies and in 30 cycles, it’s going to be 230). Therefore RT-PCR based diagnosis can detect the infection in very early stages.

The detection of the viral RNA or genes occurs along with the amplification process in a machine designated for RT-PCR. The detection is achieved using short synthetic DNA called primers. To identify a specific sequence, two primers are used which bind at the two extreme ends of the sequence and direct its amplification. For SARS CoV-2 detection, the primers used specifically bind to the ends of the gene sequences in the cDNA that are unique to the virus.

An additional component of the reaction is another unique DNA called probe. The probe also binds to the viral gene sequences in the cDNA at a position close to, but different than the primers (within the region of amplification). It has a fluorescent tag attached to it, which remains in an inactivated/quenched state. When the viral gene sequence specified by the primers gets amplified, simultaneously, the probe gets detached from it.  This releases the fluorescent tag from the probe which is detected as the signal in the RT-PCR machine using lasers (Figure 2). The detachment of probe occurs as the polymerase runs through the sequence; the polymerase can be visualized as a ‘Pac-Man’ eating away things in it way. It is for this reason that this particular RT-PCR method is called the TaqMan method, ‘Taq’ refers to the polymerase. Only patient samples that contain the virus would give a signal since the primers and probes used are specific for the viral genes. The signal is directly proportional to the viral load.

Figure 2: The fluorescence detection method: The fluorescence from the probe allows us to follow the amplifcation in real time, hence this is also known as realtime-PCR

In addition to Covid-19, the RT-PCR method of diagnosis is also routinely used for several viral infections, notably H1N1 (influenza) virus and dengue virus. The test heavily relies on the specificity of the primers and probes. In order to design specific primers and probes, the sequence of the viral genes must be known. It is commendable that the scientific community was not only able to identify the cause of a new disease, but also decipher the genetic sequence of the novel virus and design diagnostic tests; all in less than a few months of the disease emergence. Several people are still working day and night to develop quicker and inexpensive diagnostic tests.

Annapoorna P K and Santosh K Kuncha, CCMB & Dr Ramesh Byrapaneni MD, Endiya Partners: 

Opinions expressed solely are our own and do not express the views or opinions of my organizations we are associated with.