Calibration of RT-PCR test to establish value of cycle threshold
Dear Public Health England,
Please first of all refer to this paper , published by the European Journal of Clinical Microbiology & Infectious Diseases on 27 April 2020.
This paper purports to establish a significant relationship between viral RNA load detected using RT-PCR and culture positivity in SARS-CoV-2 patients.
As you can see from the fig 1 in the paper as the number of cycles required in the PCR test to measure a positive result increases, the observed cytopathic effects of the same sample inoculated in to a cell culture, decreases. At around 35 cycles the percentage of positive culture measured in the sample drops to zero.
In the paper it states
"One limitation of our work is that it cannot be extrapolated to other hospital centers since they use different systems of sample transport, of RNA extraction, and of PCR with different primers and probes; i.e. it has been suggested that sensitivity of amplification based on Gene E detection would be less sensitive than ORF1ab or N genes. We propose that each center perform its own correlation between culture results and viral RNA load from patients samples."
Can you confirm that all PHE laboratories and NHS laboratories that have been conducting RT-PCR testing have performed the type of calibration mentioned above to determine the Ct value above which the RT-PCR test can no longer detect any viral product?
If not can you tell me what the Ct values for each and every laboratory are and how they have been determined?
As you can imagine it is critical that the Ct value for every laboratory has been correctly and accurately calibrated otherwise the RT-PCR tests are worthless. Furthermore, it is clear from figure 1 that at 27 cycles the test gives 50 percent false positives.
It is also clear from this analysis that the RT-PCR test CANNOT be used as a diagnostic test for infection unless the Ct value is around 5. Of course even then this would only prove that a specific strand of RNA (ignoring the erroneous detections attributable to other strands of similar composition ) purportedly belonging to a virus called SARS-CoV-2 had been detected. It is not viral isolation. But that is another story.
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