Is RT-dPCR technology reliable for detecting COVID-19 infection?

ABOUT RT-PCR technology

In this post, we will discuss whether RT-PCR technology is reliable for testing a suspect with COVID-19 or not through properly analyzing the available facts.

What is RT-PCR technology?

Reverse Transcription-Polymerase Chain Reaction is a lab technique used by Bio-chemists to generate billions of copies of a complementary DNA generated through reverse transcription of RNA of a virus. You might be wondering how does this method help in detecting a particular disease within an individual. Well, for that you need to understand what viruses are and what is their function.

Function of a Virus

A virus is a microscopic package of genetic information surrounded by a lipid bilayer. Genetic information is in the form of RNA along with reverse transcriptase(an enzyme which converts RNA into a complementary DNA or false DNA), Integrase(an enzyme which binds complementary DNA to the nucleus of the living cell) and protease(the enzyme which cuts up the protein for the formation of a new virus).

What the virus does is it feeds its complementary DNA to the nucleus of the cell and converts that healthy cell into a virus manufacturing house.

Applying real-time RT-qPCR technology to detect a virus strand within an individual.

In real-time Reverse Transcription-quantitative Polymerase Chain Reaction, Biochemists collect a sample from the infected part of the human body(nose or mouth) and the sample is purified from any form of impurities by treating it with suitable reagents. After this, Biochemists treat the sample with a reverse transcriptase enzyme to convert RNA(if present) into complementary DNA or false DNA. Once the process of Reverse Transcription is complete, the sample of complementary DNA is intercalated with a fluorescent dye, and PCR is carried out as follows.

Step (1.) The complementary DNA is heated and separated at high temperatures resulting in Nucleic Acid Denaturation.

Step (2.) The temperature is lowered and primers(Primers are short-single strand DNA fragments that are opposite in terms of the sequence of the targeted DNA strand which was separated in step 1) bind with the single strand of DNA produced in step 1.

Once these steps are complete two new copies of the original complementary DNA are obtained. These 2 new strands become the base for DNA polymerase(an enzyme that catalyzes the conversion of nucleotides(building blocks of DNA) into DNA). Thus, DNA polymerase initiates a chain reaction that will create billions of copies of the complementary DNA. The complementary DNA is amplified until the fluorescent dye signals that the virus is present. The cycle threshold(number of amplification cycles required to reach that signal level) is used to calculate the number of target molecules originally present based on a standard curve. (relative quantification)

Though this method might seem promising, it comes with a lack of accuracy. Through experimenting with this technology, biochemists found that RT-qPCR is 30~60% accurate.

Applying real-time RT-dPCR technology to detect a virus strand within an individual.

real-time Reverse Transcription-digital Polymerase Chain Reaction is almost similar to real-time Reverse Transcription-quantitative Polymerase Chain Reaction though it does not employ a fluorescent dye for detection of a virus strand. Instead, it divides the sample into thousands of independent PCR reactions and labels them as positive or negative for amplification.

If the result is positive, then the given sample is amplifiable and hence, the virus is actually present within the individual from whom the virus sample is taken.

If the result is negative, then the given sample is not amplifiable and RNA of the virus is absent in the given sample, hence the individual is not infected with the target virus.

This process of not using a fluorescent dye simply increases the accuracy index of detection up to 90~95% as there is no requirement of using a standard curve. Also, the quantification is absolute in RT-dPCR technology and not relative, unlike RT-qPCR.

Though this can be considered a drawback as the accuracy is not 100%. One person out of 100 healthy individuals will be pointed out as positive by the mechanism of RT-dPCR.

Inference

I would say that the use of the RT-dPCR machine is not reliable for testing Coronavirus patients as there is a possibility that even a healthy individual will be dragged to the quarantine zone for COVID-19 patients.