Advancements in molecular biology have improved medical diagnosis by leaps and bounds. We can now make quicker and definitive diagnoses for conditions ranging from infertility to cancer. In the past year, there has been a massive and urgent need for rapid testing for COVID-19. The two widely used methods for diagnosing COVID-19 are antigen testing and PCR (Polymerase Chain Reaction).
Antigen Testing
The most common antigen test appears like the home pregnancy test. An antigen refers to a protein displayed on the surface of the virus. The COVID-19 antigen tests rely on the specific binding of an antigen in the sample to antibodies. These are immobilized in the spots where the indicator lines appear. Secondary antibodies bind to the antigen too and fluoresce or change color as a response. This allows detection. The degree of color change or fluorescence can be used to measure antigen levels in the sample.
In the COVID-19 antigen test, the sample is taken from the patient’s upper respiratory tract, often with an oropharyngeal or nasopharyngeal swab.
PCR Testing
PCR testing allows detection of a much lower initial viral load by making millions of copies of the sequence(s) in sample’s genetic material. These copied sequences can help in diagnosing a patient as being positive (present) or negative (absent).
In the case of COVID-19, the genetic material is RNA or Ribonucleic Acid. It is less stable compared to Deoxyribonucleic Acid (DNA).
To perform the test, one nucleic acid needs to be converted tothe other, which is achieved with the help of the reverse transcriptase enzyme. The enzyme makes a copy of the RNA from the DNA. Once that complementary DNA, or cDNA, is obtained, the PCR can be carried out.
The main components are the Taq DNA polymerase, the template, the primers, and loose nucleotides in the form of Deoxynucleoside triphosphates (dNTPs). You may think of the polymerase as a piece of machinery that assembles new segments of DNA. Just like machinery, the polymerase needs instructions on where to begin and end the sequence of the new strand. The template is the cDNA created from the sample which provides the sequence of the new strand.
You probably know about the double helix structure of DNA with two complementary strands of linked nucleotides. If one strand is present, the polymerase can produce the other strand. Primers are short DNA segments with the opposing sequence of base pairs on the template strand. dNTPs are single nucleotides which are linked together by the polymerase into the final product.
PCR involves heating and cooling the mixture to –
- Separate the complementary strands
- Adhere the primers
- Allow the polymerase to replicate the segment of interest.
Repeating this process allows the original strand to be exponentially amplified.
On average, PCR tests undergo 40 of these loops, creating over one trillion copies of the initial strand. This degree of amplification allows the virus to be detected even at extremely low concentrations. As a result,a high degree of accuracy can be achieved with small sample size.
In a process known as quantitative PCR, or qPCR, the virus is detected through the use of fluorescent markers attached to a probe. This probe is a short segment of cDNA that adheres to the template and is destroyed when the copy is made. Upon destruction, the marker fluoresces. A detectable level of fluorescence shows that the cycle threshold (Ct) has been reached and can be used to calculate the concentration of the original template in the sample.
Is PCR better than Antigen Testing?
Antigen testing may give a false negative as it requires thousands of virions to be present for a positive result. This does not allow early detection, when the viral load in the sample is too low to register. False-positive tests are also possible with antigen-based tests if a related viral species binds in place of the SARS-CoV-2 virus, causing the color change or fluorescence.
While it may take a little longer to get PCR results, the higher sensitivity ensures more reliable results.
MagBio Genomics is an industry leader in magnetic bead-based products for isolation of RNA which is a crucial step that needs to be performed before PCR testing. If you are looking for Viral RNA Extraction Kits or DNA/RNA Extraction kit manufacturers in USA, call MagBio Genomics at (301) 302-0144 OR reach out at info@magbiogenomics.com.
