PCR TECHNIQUE DECODED: Nucleic Acid Amplification Tests (NAAT)

Real-time reverse transcription-polymerase chain reaction (rRT-PCR) is the current gold standard for diagnosing suspected cases of COVID-19. rRT-PCR is a nucleic acid amplification test (NAAT) that detects unique sequences of the virus that causes COVID-19 (SARS-CoV-2) in respiratory tract specimens. The N, E, S, and RdRP are the viral genes currently targeted.

 A validated diagnostic workflow for detecting SARS-CoV-2 has been recently published by Corman and colleagues (PMID: 31992387), as follows: (a) First line screening: E gene, (b) Confirmatory screening: RdRP gene, and (c) Additional confirmatory screening: N gene.

SPECIMEN COLLECTION: 

CDC recommends collecting and testing an upper respiratory specimen for initial diagnostic testing for SARS-CoV-2. The following are acceptable specimens:

  • A nasopharyngeal (NP) specimen is collected by a healthcare professional.
  • An oropharyngeal (OP) specimen is another one collected by a healthcare professional.
  • A nasal mid-turbinate swab collected by a healthcare professional or by a supervised onsite self-collection (using a flocked tapered swab)
  • An anterior nares (nasal swab) specimen collected by onsite or home self-collection (using a flocked or spun polyester swab) or healthcare professional.
  • Nasopharyngeal wash/aspirate/aspirate or nasal wash (NW) specimen collected by a healthcare professional.
  • When under certain clinical circumstances (e.g., those receiving invasive mechanical ventilation), a lower respiratory tract aspirate or bronchoalveolar lavage sample should be collected and tested as a lower respiratory tract specimen.

SPECIMEN STORAGE

 After collection specimens should be stored at 2-8°C for up to 72 hours. Store specimens at -70°C or below if a delay in testing or shipping is expected.

A swab is taken and it is placed immediately into a sterile transport tube containing 2-3mL of either viral transport medium (VTM), Amies transport medium, or sterile saline and sent to a laboratory for further analysis. Analysis needs to take place within a few days of the sample being taken.

DNA makes up our genetic material and that of some types of viruses. But the virus which causes COVID-19, SARS-CoV-19, contains single-stranded RNA. As the PCR tests can only make copies of DNA, we need to convert the RNA into DNA first.

The virus RNA is extracted from the swab sample. It then needs to be purified from the human cells and enzymes which may otherwise interfere with the PCR test.

The purified RNA is mixed with an enzyme called reverse transcriptase. This enzyme converts the one-stranded RNA to double-stranded DNA so that it can be used in the PCR test.

The virus DNA is then added to a test tube to which the following are also added:

  • Primers: these are short sections of DNA designed to bind to characteristic parts of the virus DNA. Because of this they won’t bind to DNA which isn’t from the virus.
  • Nucleotides: these are the building blocks that make up DNA.
  • A DNA-building enzyme: this makes copies of the DNA.

A PCR analyzer heats the mixture. This causes the double-stranded DNA to unravel, and the primer can then bind to the DNA as it cools. Once the primers have bound to the DNA, they provide a starting point for the DNA-building enzyme to help copy it. Until millions of copies of the DNA have been created this process continues through repeated heating and cooling.

This explains how PCR amplifies the virus's genetic code, but not how it’s detected. This is where fluorescent dyes, added to the test tube while the DNA is being copied, come in. They bind to the copied DNA, which boosts their fluorescence, making them give off more light. It’s this light that allows us to confirm the presence of the virus.

The fluorescence increases as more copies of the virus DNA are produced. If the fluorescence crosses a certain threshold which is set above the expected background levels, the test is then considered positive. If the virus wasn’t present in the sample, the PCR test won’t have made copies, so the fluorescence threshold isn’t reached — the test is negative.