SARS-CoV-2 real-time reverse transcription polymerase chain reaction (RT-PCR) tests, can be inaccurate in two ways, as can all diagnostic assays. A false-positive result mistakenly identifies a person as infected, resulting in unnecessary quarantine and contact tracing. False-negative results are far more problematic because infected people may not isolate themselves and can infect others.
The use of full process controls in SARS-CoV-2 real-time RT-PCR assays increase their accuracy by identifying false positive and false negative samples. When the expected result for a control is obtained, the test is confirmed as working within specifications. When a control does not give the expected result, the test in question is shown to not meet expected performance.
Assess SARS-CoV-2 Controls Prior to Assessing Samples
Prior to assessment of sample test results for SARS-CoV-2 RNA detection, the positive and negative controls must be examined and confirmed to be valid and acceptable. Additionally, the internal control also needs to be assessed in both the positive and negative controls. If any of these controls are not valid, the sample results should not be interpreted.
Details describing the cut off values for SARS-CoV-2 real-time RT-PCR assays can be found in their manuals. The cutoff values are based on the assays Limit of Detection (LOD). An assay with a low LOD and cut-off value is more likely to detect SARS-CoV-2 in samples with low viral loads which may be missed by assays with higher LODs. For more information about assay sensitivity and limits of detection read the blog post, The Importance of Sensitivity in SARS-CoV-2 Real-Time RT-PCR Assays.
Invalid test results are obtained if the positive control or internal controls do not amplify or if the negative control does amplify. If an invalid test result is obtained, the sample needs to be re-tested either by extracting fresh RNA from the existing sample or by collecting a new sample from the patient.
Inconclusive SARS-CoV-2 Testing Results
SARS-CoV-2 real-time RT-PCR assays typically assess the presence of two or more SARS-CoV-2 genes in a sample. The strength of the signals for these gene targets depends upon the viral load of the patient. The presence of one gene target within the defined Ct range for SARS-CoV-2 is all that is needed to determine if a sample is positive. If one gene target is detected at the high end of the measurable range and the other targets in the assay are not detected, the sample needs to be re-tested. If inconclusive results are obtained a second time, the sample needs to be re-tested either by re-extracting fresh RNA from the existing sample or by collecting a new sample from the patient and testing the new sample.