The Clinical application of RT-PCR Diagnostic Techniques for SARS-COV2 detection

Alex D'Souza, Healthcare Tech Outlook | Friday, June 11, 2021

A bright future awaits PCR. Modernized PCR shortened the time to diagnosis while reducing the number of false positives.

FREMONT, CA: It was imperative to develop reliable diagnostic tools to identify SARS-CoV-2 infection in immune-naive individuals. The extensive testing capacity built for SARS-CoV-2 detection and diagnosis has proven essential in identifying and isolating infected individuals, which has halted the spread of the virus. PCR has proven to be just as effective as the gold standard for some test outcomes, like identifying bacteria in infectious keratitis. In addition, Broad-range PCR allows for distinguishing among the different microbes. Below is a more in-depth explanation of RT-PCR.

Real-time RT-PCR for SARS-CoV-2

Real-time RRT-PCR test performed in singleplex or multiplexed formats (multiple targets in a single well). First, a clinical sample control (RNase P, RNP) is set up to test the sample quality. cDNA is extracted from nasopharyngeal and oropharyngeal swabs, reverse transcribed, then amplified several times in a thermocycler machine. In conventional PCR, infectious disease is typically detected. Nonetheless, since PCR amplification processing is required after amplification, the approach is not practical for diagnosing SARS-CoV2. In addition, the PCR technology eliminates the post-amplification endpoint analysis and the amplification that is monitored in real-time.

Real-time PCR principle

Denaturing, annealing and polymerization occur during each PCR cycle. Untangling the helix occurs at a temperature of about 90 to 95°C. Primers specifically bind to complementary single-stranded DNA during annealing at 55°C. Finally, the polymerase reads the template strand and matches it with the complementary strand, resulting in 2 new helices, made up of the original strand and the complementary strand. Cycles doubled the amount of targeted genetic material. The DNA sequence is replicated several times at the end of the PCR procedure. The two sets of identical electrical charge and molecular weight will cause the bands to join together when applied to an electrophoresis gel.

Pros of SARS-CoV-2 RT-PCR:

Increased sensitivity and specificity to culture and staining
It is a versatile technique known by many medical teams.
Quickly performed in 4-8 hours.
Many samples can be combined in 96-well microtiter plates.
Positive and negative controls help to reduce the possibility of false positives and negatives in the PCR tests.
SARS-CoV-2 has tested and validated different target genes. As well, the suitable kit is selectable, depending on the requirements.

Cons of SARS-CoV-2 RT-PCR:

Tech expertise is needed to do and interpret the test
All reagents are to be stored and transported at -200 degrees Celsius.
The kit sensitivity and specificity vary. Results might vary, depending on the human error caused by interference.
It takes many steps to complete.
Quality control and instrument calibration are critical for accurate and validated results.
The extraction and PCR process requires costly consumables and advanced equipment.