I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor screening (which are similar, but different enough to require independent validations). The PCR step is sensitive enough to detect a roughly a single gene copy per reaction, but not always with a sub 40 Ct (which may or may not be an issue).
You see, the protocol is built around the primer/probe sets first published by the US CDC, so I initially adopted the criteria in the CDC EUA protocol, which stipulate a Ct value < 40.00 to classify a target as positive. This means is that amplification with a Ct value of 39.99 is classified as positive, while a nearly identical amplification curve with a Ct of 40.01 is classified as negative. Looking at similar NATs for in vitro diagnostics, most use this same cutoff. It seems fairly common with tests for other pathogens too.
For a little background, using a cutoff like this reduces the potential for subjective classification errors between users by providing a qualitative, binary interpretation for an ostensibly quantitative data readout. The trade-off is that an improperly assigned cutoff can result in systematic classification errors that are universal among all users. Anyone who has used one of the protocols based on the CDC probes has noticed that the two primer/probes sets targeting the SARS-CoV-2 viral genome do not perform equally well. It's common for test results to be classified as "inconclusive" when one viral target is positive, but the other is "negative" because of a Ct value just above the 40Ct cutoff. (I've already tweaked my protocol to reduce this kind of error, but could not eliminate it completely).
As I am writing this up for publication, I'm wondering if it would be better (more honest) to recommend a Ct cutoff that reflects the empirical range of Ct values in my validation studies, as opposed to keeping it at some seemingly arbitrary round number (even if it is almost universal among similar tests). Doing so would not change any of the major findings I report (sensitivity, specificity, etc.), but would change some of the numeric values. More importantly, I think it could help prevent misclassification of late-amplifying samples as negative.
Does anyone know of a biological or technical reason why this cutoff should not exceed a Ct of 40? Maybe this is just some convention that is blindly carried forward; or maybe there's some quirk of TaqMan chemistry to justify this value that I'm just not aware of.