Failures of the real-time RT-PCR for 2019 novel coronavirus test

a reply to: Teikiatsu

Thank you for your insight. I had my suspicions and you pretty much confirmed it.

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?

Thank you for your valued insight.
A star for you and an applause from me.

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.

Thank you Teikiatsu, and your wife Ketsuko, for your valued insight.
A star for you and an applause from me.

a reply to: Violater1

Three people on a cruise liner off Japan tested positive for coronavirus, bringing the total number of confirmed cases from the ship to 64, Japan’s health ministry said.

Royal Caribbean Cruises Ltd (RCL.N) on Friday banned “any guests holding Chinese, Hong Kong or Macau passports, regardless of when they were there last” from boarding the company’s ships www.reuters.com...

A 60-year-old American has died of the new coronavirus, the first confirmed non-Chinese death of the illness.

a reply to: Fowlerstoad

Hey, I am still thinking about this, especially your quest for 'faster results'.

I wonder - why not look for the virus's RNA genetic material initially? Maybe find a short segment of viral RNA that is unique, but as short as feasible, and look for that sequence with a RNA or DNA probe?

I don't know how you can close the window of time between when a person is infected and they start creating enough viral particles to turn up the viral RNA in any given sample … that is reality, and cannot ever be made zero percent, but looking for the viral RNA sequences may help, because viruses are not perfect, and when infected cells die, all this viral RNA drains out into the surroundings. IT seems maybe that would become detectable before the other viral products? Just thinking still.

a reply to: toysforadults

Well, then … you are spamming the thread with a useless response. What is YOUR point? Hahah

a reply to: Fowlerstoad
Coronavirus outbreak: Death toll surpasses total from SARS outbreak
Coronavirus deaths: Another 89 people died in China's Hubei province on Saturday, bringing the total death toll around the world to at least 813. The number of confirmed cases has risen to at least 27,100 in Hubei province alone, with the global number of infected now at more than 37,000 -- the vast majority in mainland China.
www.cnn.com...

originally posted by: Fowlerstoad
a reply to: toysforadults

Well, then … you are spamming the thread with a useless response. What is YOUR point? Hahah

Hes spamming all the Coronavirus threads with nonsense, and just poking people to make them irritated, I dunno what his angle is other than trying to derail.

As to this thread, therre is a wealth of information here, and i find it interesting that with all the news sources we have and their endless ability to tap resources, WHY no one is looking into this further and reporting it.

That should give us a supremely eye opening idea of where our media and gov sit.

a reply to: Violater1
From Yahoo News: news.yahoo.com...

"Coronavirus test kits shipped from the Centers for Disease Control and Prevention to labs across the country and around the world last week have had problems detecting the virus, the CDC said Wednesday.

During a briefing with journalists, the CDC’s Dr. Nancy Messonnier said some labs were getting “inconclusive” results from the test. As such, the test will need to be fixed.

The problem was found during initial routine run-throughs with the test to make sure it was working correctly before using it to diagnose COVID-19, as the illness is now called, in actual patients.

The issue with the diagnostic test has not been reported in all states. The Illinois Department of Public Health said Wednesday it had not experienced trouble with its test kits, and is able to move forward with testing for the coronavirus. It’s unclear how many of the test kits shipped were flawed."

This cannot be a mistake!
First the infected patient was returned to Marine Corps Air Station Miramar in San Diego, the cruise ship in NJ was allowed to release people.

originally posted by: toysforadults
I work in IT what does this mean it's a waste of time for me to read

Are you a moron, a shill or a troll?

Because based on that post, those are the only choices available.

smh...

a reply to: Violater1
The CDC is claiming that there was confusion in identifying the CovID-19 test tubes with the Miramir/San Diego patient that was released back into the group of isolated patients. Here is what a CDC 2019-nCov test kit looks like.

Notice the folded paperwork in the package. IT TAKES 2 PEOPLE TO SIGN-OFF ON THESE FORMS!
This is bull$h!t. If these forms are the same ones that I fill out, there was no way to make an error!

a reply to: Violater1

The U.S. Navy Hospital Guantanamo Bay, has plenty of room for expansion. Our Navy Seabees, together with the U.S. Army Corp of Engineers, can expand this area to accommodate 600 patients in a few weeks.


I have purposely omitted the USNS Comfort and Mercy. IMHO, it is out of the question.

a reply to: Violater1

Cepfeid a lab equipment company is now claiming they have a module for testing for the corona virus.

a reply to: Violater1

True perhaps but GITMO could only be a rule out kind of deal where people walk onto the transport. I'm confident of my ability to transport a critically ill pediatric patient over vast distances (its what I do in RL) , but the logistics of moving one ICU transport is staggering. Moving 600 ICU level patients patients would rule out any benefit IMHO. We have places here that can handle it without the risk

a reply to: Violater1


Wow! When did we hit 71!

a reply to: Violater1



Sometimes you just have to laugh!