What is the Ct value in the novel coronavirus PCR test?

Principle of PCR testing

What is PCR?

The PCR test is also well known as a test for the new coronavirus, but its official name is "polymerase chain reaction," which is an acronym for Polymerase Chain Reaction in English.

Polymerase" is an enzyme that synthesizes deoxyribonucleic acid (DNA), the nucleic acid that makes up genes, and the technique of using this enzyme to increase DNA is called PCR.

PCR technology is used not only to test for infectious diseases such as new coronaviruses, but also for basic research in medicine and biology, environmental testing such as water and soil quality, forensic investigation, and many other applications.

Principle of PCR

The PCR technique was invented in 1983 by American biochemist Kary Mullis, who subsequently won the Nobel Prize.
(There are many anecdotes about the invention, including the fact that the principle was conceived on a date during a drive, and that no one took it seriously at first because it was such an outlandish idea, so it is interesting to look it up if you are interested.)

DNA consists of two pairs of polynucleotide chains of four different base pairs: adenine (A), thymine (T), guanine (G), and cytosine (C).
This is the famous double helix structure. (Figure 1)
In PCR, these two connected strands are pulled apart by heat to form one strand each.
Next, the temperature is lowered and a short DNA molecule (primer) is attached to the single strand.
These primers are designed to bind specifically only to the portion of the gene sequence that you wish to amplify.
Finally, polymerase synthesizes DNA from the primer-bound portion, creating a new double-stranded DNA.
As you can see in the figure, one PCR reaction increases one strand of DNA to two.
By repeating the PCR reaction, the DNA increases 2-fold, 4-fold, 8-fold, 16-fold, and so on... 40 repetitions will result in an approximate 1 trillion-fold increase.
This reaction requires precise control of the temperature by raising and lowering it in a short period of time, and is performed using a special machine called a "thermal cycler.

図1 ：https://www.ac-illust.com/main/detail.php?id=1330013&word=DNA%E3%80%80%E4%BA%8C%E9%87%8D%E3%82%89%E3%81%9B%E3%82%93%E6%A7%8B%E9%80%A0

real-time PCR

The PCR technique itself is intended to increase DNA, but PCR that uses fluorescent dyes to examine how much DNA is being increased in real time is called "real-time PCR.

In PCR testing for novel coronaviruses, the real-time PCR technique is primarily used to detect DNA in real time.

What is Ct value and PCR

You may have heard the term "Ct value" used in PCR testing for novel coronaviruses.
The term is a bit difficult to understand, but simply put, it refers to the number of PCR cycles required for DNA to be amplified sufficiently to be detected.
In other words, if the amount of DNA present at the beginning is high, the Ct value will be low because a sufficient amount of DNA will be amplified in a small number of PCRs.
Conversely, if the initial amount of DNA is small, the Ct value will be higher because a large number of PCRs will be required to amplify a sufficient amount of DNA.

PCR test for novel coronaviruses

In addition to PCR, PCR testing for novel coronaviruses involves the process of extracting RNA of novel coronaviruses from nasopharyngeal wipes, saliva, and other specimens, and purifying it by removing impurities.
Also, although PCR has been described so far as a technique for amplifying DNA, novel coronaviruses are RNA viruses composed of RNA and do not contain DNA.
Therefore, an additional process called "reverse transcription," in which RNA is converted to DNA before PCR is performed, is also required.

The actual testing procedure can be summarized as follows: 1) collection of specimen (nasopharynx, saliva, etc.) → 2) RNA extraction from the specimen → 3) conversion of RNA to DNA (reverse transcription) → 4) real-time PCR.
Because of the many and complex procedures, the inspection can take several hours to complete. It is also an inspection that requires specialized and skilled techniques.

What the PCR test can tell us

Relationship between Ct values and disease status

Ct values can be used to determine whether a patient is positive or negative for novel coronavirus.
In Japan, many facilities consider a Ct value less than 40 to be positive.
As explained earlier, a Ct value of 40 means that the viral gene is amplified approximately one trillion times, which means that even a very small number of viruses can test positive.
However, there is no absolute indicator as to which value is considered positive if it is smaller than which value, and it varies from institution to institution and from country to country.
Another disadvantage is that the Ct value is difficult to use as a general indicator because the value fluctuates depending on the type of machine and reagents used in the test.

The Ct value on the day of onset was around 20, indicating a high viral load, but the Ct value increased as the disease progressed, reaching approximately 30 at 9 days of onset, indicating a decrease in viral load.
If the Ct value is 35 or higher, or if more than 10 days have passed since the onset of the disease, the number of viruses that test positive with a Ct value of 40 or lower is very small and therefore less infectious.

Ct value and super spreader

It is known that some infected individuals with the new coronavirus are "super spreaders," highly contagious infectious agents that have been the source of the spread of the virus to many people.
So, how can PCR testing be used to know who has such a highly infectious infection?

As mentioned above, Ct value is not an absolute standard, so there is no clear standard.
However, if the Ct value is smaller than 25, it can be a "super spreader" because the number of viruses is very high (about 18 million per ml) and even a small amount of saliva spatter contains enough virus to infect.
It has been reported that more than 30% of the patients had a Ct value lower than 25 even if they were asymptomatic, so it is very important to take basic infection prevention measures, including masks, even if there are no symptoms.

summary

The PCR test, a very important test for novel coronaviruses, amplifies and detects genes extracted from novel coronaviruses.
In addition to determining whether the test result is positive or negative, it is also possible to evaluate the strength of infection by determining the Ct value, which is related to the amount of virus.
It should be noted, however, that the Ct value is not an absolute indicator, as it varies depending on the instrument and reagents.

References

• The Japanese Society of Infectious Diseases – Concept of COVID-19 testing method and results
• J-STAGE – RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus
• Setagaya Ward – The 11th Setagaya Ward Mayor's Regular Press Conference in FY 2020