Novel detection method for COVID-19

Researchers at IIT Delhi Kusuma School of Biological Sciences (KSBS) have

developed a probe-free detection assay for COVID-19 which has been
approved by the Indian Council of Medical Research (ICMR). This assay has
been validated at ICMR with specificity and sensitivity of 100% that is the test
can correctly identify those with the disease and people without the disease.
This assay is a real-time PCR based diagnostic assay. Furthermore, unique
regions i.e. short stretches of RNA sequences in the COVID-19 / SARS COV-2
genome that are not found in any other human coronaviruses were identified
using comparative sequence analyses, providing opportunity to specifically
detect COVID-19.
Coronaviruses (SARS-Cov2) are those that only contain RNA. To detect
coronavirus in the body using real-time PCR, RNA should be converted into
DNA for which a process known as reverse transcription is carried out. In this
process, a primer is attached to the RNA strand and an enzyme called reverse
transcriptase is allowed to bind to the primer. The enzyme along with the primer
leads to the production of a complementary strand giving rise to cDNA. The
process of reverse transcription is carried out because only DNA can be copied
or amplified which is the key part of the real-time PCR. The resulting DNA
(with both human and viral genetic material) is then placed in the RT-PCR
machine.
The machine cycles through temperatures that heat and cool the mixture to
trigger specific chemical reactions that create new, identical copies of the target
sections of viral DNA. 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 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. Here, in the process of PCR,
some primers are used for amplification while others act as markers for the
detection of the virus. Researchers of the Indian Institute of Technology has
explained that this method uses primers targeting the unique regions of COVID
-19 that were designed and tested using real-time PCR where the primers bind
specifically to regions conserved in over 400 fully sequenced COVID-19
genomes. This highly sensitive assay was developed by extensive optimization
using synthetic DNA constructs followed by in vitro generated RNA fragments.
Though real-time PCR usually uses a probe with fluorescent dye as a marker for
the detection of the virus, researchers in IIT Delhi have developed a detection
assay that does not use any fluorescent probe and so can significantly reduce the
cost for the test, making it affordable. This highly sensitive assay was developed
by extensive optimization using synthetic DNA constructs followed by in vitro
generated RNA fragments. Thus, this assay does not require fluorescent probes
and can easily be scaled up. However, to detect past infections, which is
important for understanding the development and spread of the virus, real-time
PCR cannot be used as viruses are only present in the body for a specific
window of time. Therefore, other methods are necessary to detect, track, and
study past infections, particularly those that may have developed and spread
without symptoms.