What is qpcr threshold

It is critical to bypass or account for this background signal in order to glean meaningful information about your target. 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.

PCR reaction efficiency is dependent on the master mix performance, the specificity of the primers, the primer annealing temperature, and the sample quality. Perfect PCR efficiency coincides with a change of 3. To determine the PCR efficiency for each primer pair, run serial dilutions of your template with five fold dilution steps, and calculate the R 2 , a statistical measure that describes how well one value can predict another.

Run at least three replicates for each point on your standard curves. A higher replicate number is especially important for low copy number input, where variations across replicates are more likely. Assuming you have ruled out the 3 factors mentioned above, the most common causes of late C q values are:. To be certain that the variations in C q values are due to real biological changes and not technical issues, you will need to normalize your results.

Here, you compare the C q values of your sample to the C q values of several reference housekeeping genes. It is imperative to choose reference genes whose expression levels are not expected to change during your experiment. It is wise to use at least two reference genes, and bear in mind that what may be a reference gene for one study may not be suitable for another.

You can read more about the qPCR data analysis methods here. The number of cycles before the virus is detectable is known as the cycle threshold Ct. We have written previously about how PCR tests work, and how effective they are. A positive test with a high Ct value may indicate a test from someone who had a very small amount of detectable viral RNA on their initial swab, and may not be infectious or have ongoing active infection. However, there are other clinical scenarios that can result in a positive test with high Ct value in someone who may still be infectious or who may soon become infectious.

The claims in the Instagram post misinterpret clarifications released by the WHO. If the genetic material of interest is present in the sample, it is then copied again and again by heating and cooling the material in the presence of various substances. Each iteration of this is called a thermal cycle. As genetic material is amplified with real time PCR fluorescence is produced; how this happens exactly varies by PCR method, but basically involves those substances added to the test releasing fluorescent particles or becoming more fluorescent.

Eventually the fluorescence is strong enough to be detected. The number of thermal cycles required to reach this point is known as the cycle threshold. The fewer cycles required before that fluorescence is observed, the greater the concentration of viral genetic material in the original sample, roughly speaking.

Conversely, the more cycles that are required, the smaller the concentration of viral material on the original sample. There is concern that these positive tests may not represent people with an active infection, or who are most infectious. Recent posts have suggested that an announcement from the WHO in January somehow represents a change in approach, or an acceptance that there are flaws in PCR testing, to do with cycle thresholds that were not previously recognised.

Instead, it re-iterated the advice given in its September guidance and said that:. Where test results do not correspond with the clinical presentation, a new specimen should be taken and retested using the same or different [nucleic acid testing] technology.

The exact relationship between Ct value and infectivity is still being researched, and interpreting these results depends on the clinical context. It is important to remember that these tests are a snapshot in time. Therefore, a positive result with high Ct value could represent somebody with lower amounts of virus, and lower infectivity for example, somebody who has recently recovered from infection.

There are other factors which could also give a high Ct positive result even for someone who is infectious and has a high viral load, for example, if test samples are poorly collected or stored, or people who develop severe lower respiratory tract Covid infection but are swabbed from an upper respiratory tract site.