This issue is addressed by two values in real-time PCR: the threshold line and the C t value. Figure 1. The threshold level and C q value on a real-time PCR amplification curve. C q values are inverse to the amount of target nucleic acid that is in your sample, and correlate to the number of target copies in your sample. Lower C q values typically below 29 cycles indicate high amounts of the target sequence.
Higher C q values above 38 cycles mean lower amounts of your target nucleic acid. High C q values can also indicate problems with the target or the PCR set-up, as outlined later in the pitfalls section of this article. Your PCR instrument will collect fluorescence data during each cycle. The threshold level will be just above this, but at the point where your samples start moving into the exponential phase of PCR amplification. Today, computer software calculates this exact point and all modern real-time cyclers have an automatic threshold line setting.
In this way, C q values are usually consistent across replicates in real-time PCR. By the time the PCR reaction endpoint is reached, accumulated inhibitors, inactivated polymerases and limiting reagents create a lot of variation in endpoint values, and this is why conventional PCR cannot be used quantitatively.
Many factors can affect your C q values. Some differences in C q values between your samples will be due to biological events e. The most common pitfall areas are:. Fluorescence emission can be affected by pH and salt concentration in a solution. Any change in fluorescence emission will naturally change your C q values. Therefore, make sure you only use high-quality PCR components and if using homemade solutions, check the pH, and monitor salt precipitation before each experiment.
CT value and virus is inversely correlated. A patient with a low CT value indicates the virus is high in the body. If the CT value is low it indicates the virus is low in the body. CT value is determined by collecting swabs from the person, it is then converted to DNA, which is then amplified.
When a swab is collected from the person, it is then converted into DNA, which is amplified. Amplifications take different series of cycles. If the number of cycles is more CT value is more, the virus is not present in the body.
If the number of cycles is less CT value is less then the virus is present in the body. Normal CT levels indicate that virus is present in the body. Worldwide acceptance that CT value is above 35 to 40 indicates the covid virus is present in the body. Lower CT values indicate that there is no virus in the body. In the first report I do not really look at the Ct value, but for follow -up of patients in the hospital, I do consider the Ct value, as then I can decide whether to transfer the patient to the non-Covid building or not.
While that may be the obvious inference, some experts stress that some patients can have a high Ct value and yet have a very significant level of Covid infection, and vice versa.
Many factors are important in interpreting an RT-PCR test, and the results may also depend on the method of specimen collection and time from infection to collection and to analysis.
A poorly collected sample may reflect inappropriate Ct values. Besides, Ct values are also determined by the technical competence of the person performing the test, calibration of the equipment, and the analytical skills of the interpreters.
Again, Ct values may differ between nasal and oropharyngeal specimens collected from the same individual. The temperature of transportation, as well as the time taken from collection to receipt in the lab, can also adversely impact Ct values. Click here to join our channel indianexpress and stay updated with the latest headlines.
Must Read. Why is it important? Explained: What is CT value in a Covid test? The cycle threshold value emerges in RT-PCR tests for the coronavirus, and determines whether a person is positive for Covid or not.
To what extent does it correlate with viral load and disease severity? Written by Anuradha Mascarenhas Pune April 28, am.
0コメント