RT-PCR Testing for SARS-CoV-2
RT-PCR is a process that allows scientists to amplify a specific segment of RNA, such as part of the SARS-CoV-2 genome. But amplifying the target doesn't help without a method of detection. PCR based SARS-CoV-2 tests use molecules that fluoresce and emit light as viral targets are detected. The instruments and software used can generate graphs that trace the fluorescent intensity over time in each individual reaction well.
There are several different methods for detecting your target, but probe-based fluorophores are generally used in diagnostic PCR tests. This is because probe-based fluorophores are specific to the target being amplified. Other methods of detection, such as DNA binding dyes are non-discriminatory for nucleic acid sequence and have a greater dependence on amplification of target product. The difference can also be seen in the amount of background fluorescence emitted by non-target DNA in the reactions.
Despite the specificity of the probe to the target sequence of viral RNA there remains a considerable amount of background fluorescence in RT-PCR experiments. It is important for technologists to account for the level of background fluorescence when determining the results of diagnostic tests. Two critical values are used to determine the difference between background fluorescence and target amplification, The baseline threshold and the Ct value.
Baseline Threshold Value
The baseline threshold value (often just referred to as threshold value) is a fluorescent intensity constant that sits just above background levels. Because background traces never show a consistent exponential increase in fluorescence they can be fairly easily distinguished from target amplification. On a PCR amplification plot the baseline threshold value is generally depicted as a horizontal line that lies just above background fluorescence and intersects the exponential phase of a amplifying target traces. This value is the set value above which a target is considered positively detected. Most PCR software has a way of automatically generating this value, however labs are able to set specific values based on internal validation of test performance.
There are several different methods for detecting your target, but probe-based fluorophores are generally used in diagnostic PCR tests. This is because probe-based fluorophores are specific to the target being amplified. Other methods of detection, such as DNA binding dyes are non-discriminatory for nucleic acid sequence and have a greater dependence on amplification of target product. The difference can also be seen in the amount of background fluorescence emitted by non-target DNA in the reactions.
Despite the specificity of the probe to the target sequence of viral RNA there remains a considerable amount of background fluorescence in RT-PCR experiments. It is important for technologists to account for the level of background fluorescence when determining the results of diagnostic tests. Two critical values are used to determine the difference between background fluorescence and target amplification, The baseline threshold and the Ct value.
Baseline Threshold Value
The baseline threshold value (often just referred to as threshold value) is a fluorescent intensity constant that sits just above background levels. Because background traces never show a consistent exponential increase in fluorescence they can be fairly easily distinguished from target amplification. On a PCR amplification plot the baseline threshold value is generally depicted as a horizontal line that lies just above background fluorescence and intersects the exponential phase of a amplifying target traces. This value is the set value above which a target is considered positively detected. Most PCR software has a way of automatically generating this value, however labs are able to set specific values based on internal validation of test performance.
Each colored line is a trace that represents a single reaction/well on a PCR plate. All reactions produce some level of background fluorescence. However, the fluorescence in reactions with detected and amplified product begin to rise above the background levels.
Baseline Threshold Value
The baseline threshold value (often just referred to as threshold value) is a fluorescent intensity constant that sits just above background levels. Because background traces never show a consistent exponential increase in fluorescence they can be fairly easily distinguished from target amplification. On a PCR amplification plot the baseline threshold value is generally depicted as a horizontal line that lies just above background fluorescence and intersects the exponential phase of a amplifying target traces. This value is the set value above which a target is considered positively detected. Most PCR software has a way of automatically generating this value, however labs are able to set specific values based on internal validation of test performance.
Cycle Threshold (Ct) Values
Ct values have picked up several different names over the years:
The Ct value is the PCR cycle at which the fluorescent trace for a sample intersects the set baseline threshold value. In effect this is the number of cycles it takes to detect a true signal from a reaction well. Every reaction well has it's own Ct value so each PCR run will have many different Ct values. If a reaction trace does not go into an exponentially increasing phase or intersect the baseline threshold, the Ct value for that reaction reads as zero or "not a number".
Ct values are inverse to the starting amount of target nucleic acid in the associated sample. For a SARS-CoV-2 PCR test, this means that low Ct values (generally below 30 cycles) indicate high amounts of initial viral target sequence in the sample. Inversely, high Ct values (generally above 34 cycles) indicate lower amounts of viral target sequence in the sample.
Positive & Negative Results
Ct values for specific targets can be used to determine whether samples are positive or negative for the presence of SARS-CoV-2. Most PCR tests cleared for use by the FDA utilize a cycle cutoff between 35 and 45 PCR cycles. For example, the CDC developed RT-PCR test for detection of SARS-CoV-2 utilizes a cutoff of 40 PCR cycles. This means that any target with a Ct below 40 is considered positive for the presence of the target RNA.
You may wonder why Ct values don't get reported with test results. This is because most, if not all tests for the detection of SARS-CoV-2 are only approved for use as qualitative tests. This means that labs utilizing these tests are only allowed to use the data to determine a positive or a negative and not to quantify or report expected amounts of viral RNA target.
It should be noted that different tests use different targets, probes, and reaction chemistries. This means that not every test has been validated for use with the same cutoff values. Likewise, different labs may validate the tests in-house for use with slightly different cutoffs due to changes in equipment, processing, or environment (e.g. humidity or temperature). This means that while Ct values can be generalized to rough estimates of viral RNA copy numbers, the Ct values don't necessarily translate between labs even with the same PCR test.
The baseline threshold value (often just referred to as threshold value) is a fluorescent intensity constant that sits just above background levels. Because background traces never show a consistent exponential increase in fluorescence they can be fairly easily distinguished from target amplification. On a PCR amplification plot the baseline threshold value is generally depicted as a horizontal line that lies just above background fluorescence and intersects the exponential phase of a amplifying target traces. This value is the set value above which a target is considered positively detected. Most PCR software has a way of automatically generating this value, however labs are able to set specific values based on internal validation of test performance.
Cycle Threshold (Ct) Values
Ct values have picked up several different names over the years:
- Ct - threshold cycle
- Cp - crossing point
- Cq - quantification cycle
The Ct value is the PCR cycle at which the fluorescent trace for a sample intersects the set baseline threshold value. In effect this is the number of cycles it takes to detect a true signal from a reaction well. Every reaction well has it's own Ct value so each PCR run will have many different Ct values. If a reaction trace does not go into an exponentially increasing phase or intersect the baseline threshold, the Ct value for that reaction reads as zero or "not a number".
Ct values are inverse to the starting amount of target nucleic acid in the associated sample. For a SARS-CoV-2 PCR test, this means that low Ct values (generally below 30 cycles) indicate high amounts of initial viral target sequence in the sample. Inversely, high Ct values (generally above 34 cycles) indicate lower amounts of viral target sequence in the sample.
Positive & Negative Results
Ct values for specific targets can be used to determine whether samples are positive or negative for the presence of SARS-CoV-2. Most PCR tests cleared for use by the FDA utilize a cycle cutoff between 35 and 45 PCR cycles. For example, the CDC developed RT-PCR test for detection of SARS-CoV-2 utilizes a cutoff of 40 PCR cycles. This means that any target with a Ct below 40 is considered positive for the presence of the target RNA.
You may wonder why Ct values don't get reported with test results. This is because most, if not all tests for the detection of SARS-CoV-2 are only approved for use as qualitative tests. This means that labs utilizing these tests are only allowed to use the data to determine a positive or a negative and not to quantify or report expected amounts of viral RNA target.
It should be noted that different tests use different targets, probes, and reaction chemistries. This means that not every test has been validated for use with the same cutoff values. Likewise, different labs may validate the tests in-house for use with slightly different cutoffs due to changes in equipment, processing, or environment (e.g. humidity or temperature). This means that while Ct values can be generalized to rough estimates of viral RNA copy numbers, the Ct values don't necessarily translate between labs even with the same PCR test.
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