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Reason: None provided.

All PCR uses cycles. rRT-PCR cycles until it finds something or it washes out (no signal from background noise).

This is a brief description of rRT-PCR

Real-time reverse-transcription PCR (rRT-PCR) is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is using fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product generated. Fluorescent reporters used in real-time PCR include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that hybridize with PCR product during amplification. This value is usually referred to as cycle threshold (Ct), the time at which fluorescence intensity is greater than background fluorescence. Greater the quantity of target DNA in the sample, there will be significant increase in fluorescent signals earlier, yielding a lower Ct.

So in this case the Ct is wherever they get the signal strength they are looking for. It is difficult to imagine the number of false positives from this method.

From this report on problems with rRT-PCR from last year:

The DNA probes used in the CDC rRT-PCR test kits for SARS-CoV-2 assay are only about 25 bases long which does not meet the FDA recommendation for nucleic acid-based molecular diagnostics for viral disease infections where 100 contiguous bases is desirable (1). Various methods use different genes and different probes that may not be equivalent. There is a 100-fold difference in limit of detection (LoD) between some assays (2). Technical error, especially due to contamination may cause false positives. Seventy-seven professional baseball major league players initially tested positive in one lab but negative elsewhere (3) in what was deemed Lab error. Except that they had multiple sources for testing, they might have been classified as asymptomatic. We don’t know how many other persons were classified in error from this incident.

So its basically useless. They go on to say:

Originally, PCR was followed by a second step where a separation technique such as a blotting method was used to confirm that the amplified substance was correct. rRT-PCR is usually not followed by a second step. RRT-PCR is usually applied for diagnostic purposes, not for screening.

So its useful for making sure if you have disease symptoms that you have a certain virus, or a related virus, depending on the probes used since it should show up at a very low cycle threshold.

What does it mean "related virus?" Here's an example; doing a PCR with the a probe for the N-protein of SARS might find H1N1, since the sequence is similar enough that if you cycle it high enough you will likely get H1N1 regardless (since they both have similar N-proteins). You also might get a cold virus for the same reason. You also might get a bacteria that has incorporated a similar protein, or a cell in your own body that did the same last time you got a cold, etc. That is why Ct is so important here, and why rRT-PCR is so ridiculous since the Ct is basically "whatever it takes to get a signal until we are 100% sure there isn't one".

The fact that they look at multiple sequences should help reduce false positives, but without looking at the protocol and mapping the probes to all viral, human, and bacterial genomes there is no way to know how accurate it might be.

2 years ago
1 score
Reason: Original

All PCR uses cycles. rRT-PCR cycles until it finds something or it washes out (no signal from background noise).

This is a brief description of rRT-PCR

Real-time reverse-transcription PCR (rRT-PCR) is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is using fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product generated. Fluorescent reporters used in real-time PCR include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that hybridize with PCR product during amplification. This value is usually referred to as cycle threshold (Ct), the time at which fluorescence intensity is greater than background fluorescence. Greater the quantity of target DNA in the sample, there will be significant increase in fluorescent signals earlier, yielding a lower Ct.

So in this case the Ct is wherever they get the signal strength they are looking for. It is difficult to imagine the number of false positives from this method.

From this report on problems with rRT-PCR from last year:

The DNA probes used in the CDC rRT-PCR test kits for SARS-CoV-2 assay are only about 25 bases long which does not meet the FDA recommendation for nucleic acid-based molecular diagnostics for viral disease infections where 100 contiguous bases is desirable (1). Various methods use different genes and different probes that may not be equivalent. There is a 100-fold difference in limit of detection (LoD) between some assays (2). Technical error, especially due to contamination may cause false positives. Seventy-seven professional baseball major league players initially tested positive in one lab but negative elsewhere (3) in what was deemed Lab error. Except that they had multiple sources for testing, they might have been classified as asymptomatic. We don’t know how many other persons were classified in error from this incident.

So its basically useless. They go on to say:

Originally, PCR was followed by a second step where a separation technique such as a blotting method was used to confirm that the amplified substance was correct. rRT-PCR is usually not followed by a second step. RRT-PCR is usually applied for diagnostic purposes, not for screening.

So its useful for making sure if you have disease symptoms that you have certain virus, or a related virus, depending on the probes used since it should show up at a very low cycle threshold.

What does it mean "related virus?" Here's an example; doing a PCR with the a probe for the N-protein of SARS might find H1N1, since the sequence is similar enough that if you cycle it high enough you will likely get H1N1 regardless (since they both have similar N-proteins). You also might get a cold virus for the same reason. You also might get a bacteria that has incorporated a similar protein, or a cell in your own body that did the same last time you got a cold, etc. That is why Ct is so important here, and why rRT-PCR is so ridiculous since the Ct is basically "whatever it takes to get a signal until we are 100% sure there isn't one".

The fact that they look at multiple sequences should help reduce false positives, but without looking at the protocol and mapping the probes to all viral, human, and bacterial genomes there is no way to know how accurate it might be.

2 years ago
1 score