I'm collecting articles describing problems with PCR testing for COVID-19. Here I will share the best information that I found so that others can benefit too. If anyone else knows of any good sources, please share them here too.
Biologist and "myth buster" David Crowe collected excellent information on covid-19 PCR tests here: Issues with the RT-PCR Coronavirus Test
Some interesting bits:
"The PCR algorithm is cyclical. At each cycle it generates approximately double the amount of DNA (which, in RT-PCR, corresponding to the RNA that the process started with). When used as a test you don’t know the amount of starting material, but the amount of DNA at the end of each cycle will be shown indirectly by fluorescent molecules that are attached to the probes. The amount of light produced after every step will then approximately double, and when it reaches a certain intensity the process is halted and the sample is declared positive (implying infected). If, after a certain number of cycles, there is still not sufficient DNA, then the sample is declared negative (implying not infected). This cycle number (Ct) used to separate positive from negative is arbitrary, and is not the same for every organization doing testing. For example, there is a paper published that reported using 36 as the cutoff for positive, 37-39 as indeterminate, requiring more testing, and above 39 as negative. Another paper used 37 as the cutoff, with no intermediate zone. In a list of test kits approved by the US FDA one manufacturer each recommended 30 cycles, 31, 35, 36, 37, 38 and 39. 40 cycles was most popular, chosen by 12 manufacturers, and one each recommended 43 and 45."
"Implicit in using a Ct number is the assumption that approximately the same amount of original RNA (within a multiple of two) will produce the same Ct number. However, there are many possibilities for error in RT-PCR. There are inefficiencies in extracting the RNA, even larger inefficiencies in converting the RNA to complementary DNA (Bustin noted that efficiency is rarely over 50% and can easily vary by a factor of 10), and inefficiencies in the PCR process itself. Bustin, in the podcast, described reliance on an arbitrary Ct number as “absolute nonsense, it makes no sense whatsoever”. It certainly cannot be assumed that the same Ct number on tests done at different laboratories indicates the same original quantity of RNA."
"Professor Bustin stated that cycling more than 35 times was unwise, but it appears that nobody is limiting cycles to 35 or less (the MIQE guidelines recommend less than 40). Cycling too much could result in false positives as background fluorescence builds up in the PCR reaction."
"The Ct cycle number will significantly influence the number of positive tests. If the Ct was changed from 37 to 35 there would be fewer positive tests, and if changed to 39 there would more positive tests. Even if the Ct number was standardized, it would still have different meaning depending on the specific machines, chemicals and procedures used by different labs, and even within the same lab changes could still be found between different runs of samples. Without simultaneously amplifying a known quantity of ‘spiked’ RNA, it cannot be assumed that with consistent Ct numbers can be used to consistently provide a boundary between positive and negative."
Biologist and "myth buster" David Crowe collected excellent information on covid-19 PCR tests here: Issues with the RT-PCR Coronavirus Test
Some interesting bits:
"The PCR algorithm is cyclical. At each cycle it generates approximately double the amount of DNA (which, in RT-PCR, corresponding to the RNA that the process started with). When used as a test you don’t know the amount of starting material, but the amount of DNA at the end of each cycle will be shown indirectly by fluorescent molecules that are attached to the probes. The amount of light produced after every step will then approximately double, and when it reaches a certain intensity the process is halted and the sample is declared positive (implying infected). If, after a certain number of cycles, there is still not sufficient DNA, then the sample is declared negative (implying not infected). This cycle number (Ct) used to separate positive from negative is arbitrary, and is not the same for every organization doing testing. For example, there is a paper published that reported using 36 as the cutoff for positive, 37-39 as indeterminate, requiring more testing, and above 39 as negative. Another paper used 37 as the cutoff, with no intermediate zone. In a list of test kits approved by the US FDA one manufacturer each recommended 30 cycles, 31, 35, 36, 37, 38 and 39. 40 cycles was most popular, chosen by 12 manufacturers, and one each recommended 43 and 45."
"Implicit in using a Ct number is the assumption that approximately the same amount of original RNA (within a multiple of two) will produce the same Ct number. However, there are many possibilities for error in RT-PCR. There are inefficiencies in extracting the RNA, even larger inefficiencies in converting the RNA to complementary DNA (Bustin noted that efficiency is rarely over 50% and can easily vary by a factor of 10), and inefficiencies in the PCR process itself. Bustin, in the podcast, described reliance on an arbitrary Ct number as “absolute nonsense, it makes no sense whatsoever”. It certainly cannot be assumed that the same Ct number on tests done at different laboratories indicates the same original quantity of RNA."
"Professor Bustin stated that cycling more than 35 times was unwise, but it appears that nobody is limiting cycles to 35 or less (the MIQE guidelines recommend less than 40). Cycling too much could result in false positives as background fluorescence builds up in the PCR reaction."
"The Ct cycle number will significantly influence the number of positive tests. If the Ct was changed from 37 to 35 there would be fewer positive tests, and if changed to 39 there would more positive tests. Even if the Ct number was standardized, it would still have different meaning depending on the specific machines, chemicals and procedures used by different labs, and even within the same lab changes could still be found between different runs of samples. Without simultaneously amplifying a known quantity of ‘spiked’ RNA, it cannot be assumed that with consistent Ct numbers can be used to consistently provide a boundary between positive and negative."