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originally posted by: Fowlerstoad
a reply to: Violater1
Hey, I took a rest, and now I am back, and have been thinking about this thread discussion since.
The RT PCR test for this novel coronavirus has a certain sensitivity, and needs the virus to infect a person and to start replicating for a long enough time for the RNA amount to accumulate, in order to trigger the reverse transcriptase in a sample to amplify the abnormal sequences created by this novel coronavirus in order to show up as 'positive'. There is always noise, and that is the lower limit of detectability.
Unfortunately, beyond this point of the discussion, I personally lack the expertise needed to explain the molecular science of what receptors and such are involved, or the differences as they may be between SARS and this new virus, or MERS. I just don't have good knowledge of that -- in fact actually, I am learning from this thread hahahaha
But zooming back out to the larger focus picture, I think initially patients may test negative because they have not had the virus long enough for it to 'make itself detectable' by replicating its RNA, and making enough new virions to show up in any given sample.
Every type of test has a lowest detection threshold, and below that, all tests would be negative or indeterminate. This is just a test, and can be improved (hopefully). So an infected patient may initially test negative, and if you wait, and retest these negative patients who have the virus, later they can become 'Positive' because now there is enough abnormal viral RNA in your sample to detect.
So, with this new virus, POSITIVE means a lot more than initial negative results, as the test isn't yet sensitive enough to find and separate out the early not yet replicating patients. It is a work in progress?
originally posted by: Teikiatsu
Hello everyone, mt wife Ketsuko has asked me to weigh in on this thread.
I've worked 20 years in a vaccine quality control lab and while I have not run PCR directly I *have* worked with people who run these type of tests and I have reviewed and trended the test results. I'm not a expert but I can fake it. As an addendum, I have played host to visiting Chinese biologists and have a feel for their situation from at least 5 years ago.
Anyways. PCR can be a very sensitive test if done correctly. It requires the proper timing, the correct amount of reagents, correct concentration of nucleic acids, the proper timing, proper heating and cooling cycles and equipment, etc. If any part of that is not followed correctly, the cascade of biochemical reactions that gives you a proper and sensitive result will not be accurate.
PCR does one thing: it finds a specific nucleic acid sequence (RNA or DNA) and once it finds that sequence it replicates it for as many times as the replication cycle is performed correctly and for as long as there are free nucleic acids to piece together that sequence into copies. Every PCR cycle doubles the amount of sequence. Then recognition proteins with radiological or fluorescent markers are added to the final result and the amount of sequences are measured against a control value to determine how much of the original sequence was present.
PCR doesn't care where the sequence comes from. It can come from inside a cell/virus capsule or free-floating fragments from a dead cell or inactivated capsule. Reading the literature above it seems that the PCR kits themselves take great pains in their protocols to make sure that the sequences are coming from a live virus. The trick is, how are the virus samples prepared? This is where false readings can come from. If the virus is still in coated in extra material that could inhibit the sample preparation then the kits may not get to the sequences efficiently, and you get false results.
This is where my experience with Chinese biologists comes in. When we had them visit our lab, they were more interested in our lab equipment catalogs than our lab equipment. The idea of multiple vendors and disposable lab equipment was (pardon the pun) totally foreign to them. The scientists I met had very limited resources in their home country. They were lucky to have a choice in reagents, glassware, equipment, etc.... and by choice I mean 2 or 3 options. The idea of disposable plastic weigh boats for chemical powders astounded them. In their labs *everything* was cleaned and re-used (sometimes by hand, no automated/validated washers). This is a recipe for contamination and false positive or negative results. Maybe things have improved since then, but 5 years is not a lot of time to overhaul a system.
So when I hear the Chinese are getting questionable results, I'm not surprised. It's not that the technicians are necessarily bad, and it's not that the PCR kits are not up to the task. But if they are still working with second-rate older equipment or bargain basement reagents then the data they capture is going to have a lot of variability. Then add in that PCR can be twitchy and needs proper timing and properly calibrated incubation, etc... it's not a good combination.
originally posted by: Fowlerstoad
a reply to: toysforadults
Well, then … you are spamming the thread with a useless response. What is YOUR point? Hahah