COVID-19 Testing

Why Use a More Painful Nasal Swab?

COVID-19 testing has now entered our daily life, but many people still have big questions about the principles of nucleic acid testing. Why do nucleic acid testing require the use of a nasal swab? What is the reason for this? Let's take a look at it from the perspective of the principle.

The most commonly used technique for nucleic acid tests is RT-PCR (Real-time Quantitative PCR, Real-time Polymerase Chain Reaction), also called qPCR (Quantitave Polymerase Chain Reaction), which is actually a very mature technique in clinical practice. qPCR (RT-PCR) is currently the preferred method for DNA/RNA quantification in the field of molecular biology.

In contrast to conventional PCR, qPCR is a method capable of real-time quantification, which means that it can monitor the exact amount of amplified DNA in the sample in real time. However, in conventional PCR, amplified DNA can only be detected after the completion of amplification (endpoint detection).

To take COVID-19 virus as an example, the viral RNA can be used as an amplification template, reverse transcribed to cDNA, and then amplified by qPCR.

In the qPCR reaction, the PCR being amplified is labeled with a fluorescent fuel (commonly SYBR GREEN I) and fluorescence is detected throughout the qPCR process (30-45 cycles). The more DNA molecules in the sample, the faster the fluorescence increases during the PCR cycle. qPCR fluorescence signal intensity is proportional to DNA initial amount, i.e., if the more virus there is in the sample, the earlier the fluorescence will be detected in the cycle. The cycle in which the fluorescence signal can be detected is called Crossing Point (Cq for short).

P2: The picture depicts the qPCR amplification (first two cycles) occurring in a PCR tube. qPCR has different variants of the signaling molecule and different methods for its fluorescent labeling can be implemented. The two most common principles are shown in the picture. The left section shows the 5′-nuclease variant, which uses the FRET mechanism (fluorescence resonance energy transfer), where the fluorescence of the reporter fluorophore (R) is transferred to the quencher (Q) and is not emitted as long as the R and Q are together (such as TaqMan). When the two are delocalized (when the probe is removed during PCR extension by the 5′-de nuclease activity of TaqDNA polymerase), the R is free to fluoresce and then can be detected. The right section represents a qPCR variant that uses intercalation fluorescence (e.g. SYBR Green). Special intercalation dyes are used that strongly increase the amount of fluorescence emitted whenever they are inserted in dsDNA.

P3: Amplification plots for five samples (S1- S5) are shown. As the DNA in each sample is amplified, the fluorescence increases for each cycle. In the example above, sample S1 contains the highest initial amount of target DNA, resulting in the fastest increase in fluorescence. Sample S4 contains the lowest number of initial target DNA molecules, while S5 does not contain any target DNA.

In other words, the more virus there is in the nasopharyngeal swab, the easier it is to detect the new coronavirus. Therefore, in clinical practice, a nasal swab will be applied for accuracy, and the nasal cavity is generally considered to contain a higher viral load.

Thus, would you prefer to use a nasal swab or a pharyngeal swab? Tell us what you think!