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

I would appreciate comments especially from Ketsuko, Fowlerstoad and Phage.
I just flip burgers at a hamburger joint and know nothing about anything. But I have to wonder why several patients are initially testing negative only to retest positive. One example is found here www.scmp.com...
Something in the current RT PCR test is looking for a substance that the current reagent is not finding quickly enough. Here is what the current PCR is looking at: www.eurosurveillance.org...
www.who.int...

The above represent relative positions of amplicon targets on SARS-CoV ad Wuhan-CoV genome.N: nucleocapsid; ORF: open reading frame; RdRp: RNA-dependent RNA polymerase.Numbers below amplicon are genome positions according to SARS-CoV, NC_004718.
This diagram represents what the PCR is looking for.


The PCR is looking for what 6-Carboxyfluorescein (6-FAM) attaches too. 6-FAM attaches to the Oligonucleotide RdRp_SARSr-P2 and RdRP_SARSr-P1 It is the test used by the CDC and WHO. www.who.int...
6-FAM is a fluorescent dye and is membrane-impermeant, and can be loaded into cells by microinjection or scrape loading. It can be incorporated into liposomes, and allow for the tracking of liposomes as they pass through the body. A liposome is a spherical vesicle having at least one lipid bilayer.

A microfluidic based mixing of lipids, helper lipid DSPC (1), PEG-lipid (2), ionizable amino lipid DLin-MC3-DMA (3) and cholesterol(4), diluted in ethanol and mmRNA(5) diluted in acidic acetate buffer (pH 4) to construct LNPs encapsulated with mmRNA. Upon mixing in acidic condition, electrostatic interactions drive the formation of inverted micelles containing mmRNA molecules surrounded predominantly by ionizable lipid, which are then coated with PEG-lipids to form the LNPs. After LNPs formation, the pH is raised to physiological pH, leading to LNPs with a biocompatible neutral charge. Subsequently, upon endocytosis to the acidic endosome, the amino group of the DLin-MC3-DMA becomes positively charged, which allows the release of mmRNA molecules to the cytosol. b Representative transmission electron microscopy (TEM) images of LNP encapsulating fLuc mmRNA (left) and IL10 mmRNA (right). c Encapsulation efficiency as measured using RiboGreen assay. Free mmRNA concentration in Triton-permeabilized LNPs solution divided by free mmRNA concentration in intact LNPs solution. So 6-FAM attaches to FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ and identifies it as 2019-nCoV. www.nature.com...

However, this engineered virus may not purposely replicate enough helper lipid DSPC (1), PEG-lipid (2), ionizable amino lipid DLin-MC3-DMA (3) and cholesterol (4) for the 6-FAM to bind too. Until the viral load, meets the level for enough 6-FAM to bind too, it’s too late.
Perhaps a test for the targeting moiety (the specific antigen and Compliment that attaches to the epithelium of the alveoli), would reveal faster results.

Additional sources:
onlinelibrary.wiley.com...
www.statnews.com...
www.ecdc.europa.eu...
www.genengnews.com...

As always, please refer to my signature below.

a reply to: Violater1

Coronavirus: Canada reports fourth case, a patient who tested negative at first.
www.scmp.com...

“That’s’ a tasty burger!” Damn son. You done did HW.

Well for detection of a virus you usually need a certain titer or viral load to be detected. To me, this suggest that the virus is doing a lytic type reproduction and that the infected cells have not released all of its viral content.

Just a thought

Contamination of the sample or sometimes the reagents is often a problem with poor PCR results. It's hard to say without the spectro results.

a reply to: feldercarb

nah, the PCR will still work if you use the correct primers.

a reply to: Violater1

The hospital is not saying if the reason the patient was sent home had a positive or negative RT-PCR test.

I wonder why.

Lab tests are only done if a doctor orders them. If the patient was only presenting mild flu like symptoms they may not have been tested at all.

it seems like it is worse that patients being tested negative, later testing positive for the virus. on the news tonight in the Philippines they were saying a sample from a patient that was released when the sample showed negative, is now showing positive. not the patient, who has apparently already returned to China, but the very sample that was tested as negative, is now testing positive.

originally posted by: generik
it seems like it is worse that patients being tested negative, later testing positive for the virus. on the news tonight in the Philippines they were saying a sample from a patient that was released when the sample showed negative, is now showing positive. not the patient, who has apparently already returned to China, but the very sample that was tested as negative, is now testing positive.

From what I have read, I'm if wondering that the virons (baby viruses) released are initally plant based and have a (so far) undetectable plant based capsule. Then when they mature inside a cell, they infect the cell and release the lipid capsule of the mutated viruses (think Alien, like the movie) that now contain the lipid capsule. It is then detected by the 6-FAM RT-PCR.

Philippines confirms third case of novel coronavirus www.msn.com...

a reply to: Violater1

I am humbled and honored that you might care what I think, and this is a very complicated subject. I need some time, but I will respond Respect, and wow … there is a lot to consider what to say about this.

I work in IT what does this mean it's a waste of time for me to read

a reply to: Violater1

PCR should be able to amplify what it needs to, once the kinks, procedures and stress is all worked out.

It should be used as a confirmation test, not a screening test and even then it will never be 100%.

I think we will be okay over here, especially once they have a solid diagnostic algorithm that moves this test to confirmation rather than screening. It will save time and lives once this happens.

I’ve only performed PCR and it’s always a botched sterile environment or lazy methods, even bad products that delete, skip or miss stuff after the initial result. It’s extremely annoying, I’d rather inject crap with stuff or grow and kill it lol.

Plus we have better quality control standards, some techs are just really rushed in pathology departments too, we see that sometimes. The rest is beyond me, advanced genetics is at the end of my education so far.

Here is the proposed diagnostic algorithm that will eliminate problems with 99% of botched PCR by switching it to a confirmation test so they can already initiate treatment and isolation protocols.

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

I'm sorry, it was meant for people in medicine.
Similarly, if you posted a thread on IT server comparisons between TechMikeNY Server 2X 2.60Ghz E5-2660v3 10C 192GB High-End PowerEdge R730xd, Digiliant R3E116LS-NL-0640 64TB Linux Storage Server and a Dell PowerEdge R630 8 Bay SFF 1U Rackmount Server, 2X Xeon E5-2680 V3 2.5GHz 12 Core. I would be at a loss.

a reply to: TheAMEDDDoc

I'm sorry, but I see a clinical pathway and not
a study in viral resolution in identification, using Polymerase chain reaction.
HHS is opening research on a new way in determining the nCoV.
HHS Seeks Abstract Submissions for 2019-nCoV Diagnostics Development www.hhs.gov...
This maybe due to exactly what I have pointed out above.

a reply to: Violater1

They need 3 things, a screening test, a confirmation PCR test and a diagnostic algorithm. It’s good they’re funding an open solution now before it hits. It would also explain the large assortment of sampling locations they are asking for from possible patients.

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?

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.

a reply to: Violater1

At least four American evacuees from China who arrived Wednesday at Marine Corps Air Station Miramar in San Diego were hospitalized after showing signs of coronavirus, the pneumonia-like illness that began in the Chinese city of Wuhan and has since sickened more than 28,000 worldwide.
www.foxnews.com...

a reply to: Violater1

IMHO, I believe the possibility exists that they have crossed plant DNS into what I call the secondary virus capsule. Perhaps this explains why there are negative tests the first or up four results. Mean while the infected cell is host to the massive replication of the Primary virus. Then during lysis, they invade.