One word of warning, this looks like qPCR approach. It is not the number of cycles that counts, it is the number of cycles to threshold, whatever that threshold might be. If it is reached in 20-25 cycles, then yes, I am convinced it is real. Anything above 30 is probably false positive.
If you are old school, it can get confusing, because back in the days RT stood for "reverse transcription" or "real time", which are different things. These days, qPCR stands for "quantitative PCR", which is the same thing as the olden days "real time PCR".
These days they hunt for sniffles viral markers by RT-qPCR ("reverse transcription quantitative polymerase chain reaction"). Which means, viral RNA (colds are caused by RNA viruses) is reverse transcribed to cDNA (RT step), and then the amount of cDNA (which is expected to be proportional to the amount of viral RNA) evaluated by counting the number of cycles to threshold (qPCR step). Fewer cycles to threshold means more viral RNA in the original sample.
is there a way to determine if the tests referenced in the FDA pdf are PCR or qPCR?
thinking it would be a monumental error for the instructions to specify 45 cycles for PCR as that would be blatantly oversensitive based on everything else I've read, but people also make mistakes (like flubbing "redacted" text on govt documents where you could copy/paste into notepad and presto change-o it's unredacted)
The protocols shown specify qPCR. Then again, the only difference between PCR and qPCR is that in PCR you measure the amount of DNA at the end of the reaction (after, say, 25 cycles). In qPCR, you measure the amount of DNA after each cycle (that requires a much fancier machine). The consequence of those differences is that, by counting the number of cycles it takes to get to some threshold amount of DNA, and by comparing that number of cycles to threshold (CTT) to CTT for a known standard under the same conditions, you can estimate the amount of DNA of interest in the original sample.
As I commented earlier on this board, PCR (q or not) simply cannot work correctly at 45 cycles. Either your procedure is perfectly optimized and you are picking up medically insignificant amounts of viral RNA, or your procedure sucks and you are picking up false positives. All intermediate scenarios fall into the second category, because if you have to go 30+ cycles to threshold, then you have poorly optimized sample collection, or poorly optimized amplification. Or your threshold is set way too high.
By the looks of it, yes.
One word of warning, this looks like qPCR approach. It is not the number of cycles that counts, it is the number of cycles to threshold, whatever that threshold might be. If it is reached in 20-25 cycles, then yes, I am convinced it is real. Anything above 30 is probably false positive.
thank you!
is RT/Realtime pcr same or different from qPCR?
If you are old school, it can get confusing, because back in the days RT stood for "reverse transcription" or "real time", which are different things. These days, qPCR stands for "quantitative PCR", which is the same thing as the olden days "real time PCR".
These days they hunt for sniffles viral markers by RT-qPCR ("reverse transcription quantitative polymerase chain reaction"). Which means, viral RNA (colds are caused by RNA viruses) is reverse transcribed to cDNA (RT step), and then the amount of cDNA (which is expected to be proportional to the amount of viral RNA) evaluated by counting the number of cycles to threshold (qPCR step). Fewer cycles to threshold means more viral RNA in the original sample.
I understood some of those words
not in the medical field at all
is there a way to determine if the tests referenced in the FDA pdf are PCR or qPCR?
thinking it would be a monumental error for the instructions to specify 45 cycles for PCR as that would be blatantly oversensitive based on everything else I've read, but people also make mistakes (like flubbing "redacted" text on govt documents where you could copy/paste into notepad and presto change-o it's unredacted)
The protocols shown specify qPCR. Then again, the only difference between PCR and qPCR is that in PCR you measure the amount of DNA at the end of the reaction (after, say, 25 cycles). In qPCR, you measure the amount of DNA after each cycle (that requires a much fancier machine). The consequence of those differences is that, by counting the number of cycles it takes to get to some threshold amount of DNA, and by comparing that number of cycles to threshold (CTT) to CTT for a known standard under the same conditions, you can estimate the amount of DNA of interest in the original sample.
As I commented earlier on this board, PCR (q or not) simply cannot work correctly at 45 cycles. Either your procedure is perfectly optimized and you are picking up medically insignificant amounts of viral RNA, or your procedure sucks and you are picking up false positives. All intermediate scenarios fall into the second category, because if you have to go 30+ cycles to threshold, then you have poorly optimized sample collection, or poorly optimized amplification. Or your threshold is set way too high.
awesome - THANK YOU!
You're welcome!