COVID-19 appeared at the end of 2019 and swept across the world. By the end of May of this year, the total number of confirmed cases in Taiwan had reached 2,032,983 and the number of confirmed cases in a single day had climbed to more than 80,000. Meanwhile, terms like “PCR” and “Ct value” have made a frequent appearance in our lives. We will start with the principles of PCR, introduce what is meant by Ct values, and explain the relationship between Ct values and viral load as well as common ways in which these terms can be confused and misused.

Introduction to the Principles of Real Time PCR
Compared with traditional reverse transcriptase PCR (RT-PCR), real-time quantitative polymerase chain reaction (also known as Real Time PCR, and hereinafter referred to as “qPCR”) is based on the principle of PCR amplification to amplify the cDNA template. The difference is that Real Time PCR uses a specific primer and probe to generate a fluorescent signal during the reaction, and instant quantitative results are achieved with fluorescent detection technology equipment. When the reaction is completed, the test results can also be presented immediately.

The calibration fluorophores commonly used in qPCR can be divided into two types: non-specific (SYBR Green) and specific (TaqMan probe). Clinical testing is mainly based on TaqMan probe. The TaqMan probe is a synthetic oligonucleotide labeled with different fluorescent substances at each end. The fluorescence at the 5' end is the reporter (R) and the fluorescence at the 3' end is the quencher (Q). When the probe is in the free state, the interaction between the reporter and the quencher masks each other's fluorescent signals. When the probe is hydrolyzed as the cDNA replicates, the reporter is separated from the quencher, and the quencher loses its effect of masking the reporter, so that the reporter's fluorescent signal can be detected (Figure 1).

Figure 1: Schematic diagram of TaqMan action

Ct Value Calculation and Meaning
Since DNA constitutes semi-preserved replication, the reaction is amplified to the N power of 2 for the target template. Each instance of virus replication constitutes one cycle; and “Ct value” is short for cycle threshold value. When the qPCR reaction is performed, the target nucleic acid template in the patient specimen continues to amplify at a rate of 2 to the power of 2 (Figure 2). At the same time, the specific gene fragments of the virus are detected with the specific TaqMan probe. Detection can occur when the amount of nucleic acid is amplified beyond the set threshold/cut-off. The Ct value at this time represents the number of times that the virus has been replicated. When the Ct value is 25, for example, this means that it has gone through 25 cycles and in turn indicates that the virus has been amplified by 2 to the power of 25 before detection occurred. Therefore, when there is a higher amount of virus in a patient's body on a given day, replication needs to occur fewer times and the Ct value is also lower; and the reverse is also true.


Figure 2: Ct value calculation diagram (Ct = n; RFU = 2n)

Typically, qPCR reactions run 40-45 cycles, but how much does it take for a Ct value to be considered positive? It depends on the limit of detection (LoD) and threshold/cut-off of the reagent.
The Ct threshold of the GB SARS-CoV-2 Real-Time RT-PCR kit (4PCO052E) is 37 and the limit of detection (LoD) is 1,000 copies/ml, representing an extremely low viral load in the patient's body. The virus would need to be amplified by 2 to the power of 37 (Ct = n, copies number of viral = 2n) for detection using the GB SARS-CoV-2 Real-Time RT-PCR kit.
By understanding the principles behind the Ct value, we can get more information. For example, it is speculated that it is related to the severity of the disease, mortality rate, infectivity, and so on. Most people might intuitively think that the lower the patient's Ct value, the higher the viral load; and that symptom severity and infectivity would also be relatively high. But in fact, this phenomenon is not absolute in clinical practice. This is because Ct value is not an absolute value, and it will be affected by various factors such as sampling method, specimen type, reagent quality, and the time of sampling.

Ct value is certainly an important basis; in the current medical decision-making of COVID-19, however, Ct value is just one of the indicators for reference when a patient is confirmed to be positive. Physicians will still use the patient's symptoms as the primary basis for decision-making.

In addition to the limit of detection of the reference reagent itself, governments around the world are also easing Ct value quarantine criteria as the daily number of confirmed cases rises. If the Ct value associated with release from isolation is set higher, it will be more rigorous; and this practice is more suitable when the number of confirmed cases is small and it is necessary to prevent the spread of the virus. However, when the number of infected people is too high and too many medical resources are consumed, consideration must be given to retaining sufficient medical resources for critically ill patients as well as how to strike a balance between pandemic prevention and economic activity.

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Rabaan AA, Tirupathi R, Sule AA, et al. Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19. Diagnostics (Basel). 2021