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Questions for Native and Coronavirus discussion Thread

Californication

Varsity
10 Year Member
CA is really having trouble getting swabs, sample transport media and RNA extraction kits for the diagnostic tests. Testing is up to nearly 100,000 per day, but that is nothing to where it needs to be according to CAs plan to relax shelter at home.
I misspoke yesterday regarding CA's testing. we are currently at 16,000 tests per day, with swabs being the major issue to ramp up. CA is capable of completing 100,000 tests / day based on lab space, equipment and personnel.
 

FeelLikeAStranger

How are ya now?
15 Year Member
I misspoke yesterday regarding CA's testing. we are currently at 16,000 tests per day, with swabs being the major issue to ramp up. CA is capable of completing 100,000 tests / day based on lab space, equipment and personnel.

The FDA is now allowing some additional sample types and also swab types to be used across the board. Rutgers DNA Core got an EUA modification to use saliva samples recently, which would allow for easier sample collection, e.g. at home, without extra PPE. I read an article today about some labs going to “direct” amplification techniques without RNA extraction. One of the studies was about 10% false negative, which isnt great. Nasal swabs arent probably the best sample for that, due to the variability.
 

Native

ToungeInCheek since 2010
Staff member
10 Year Member
The FDA is now allowing some additional sample types and also swab types to be used across the board. Rutgers DNA Core got an EUA modification to use saliva samples recently, which would allow for easier sample collection, e.g. at home, without extra PPE. I read an article today about some labs going to “direct” amplification techniques without RNA extraction. One of the studies was about 10% false negative, which isnt great. Nasal swabs arent probably the best sample for that, due to the variability.
Direct amplification is great but never consistent. Especially with bacteria. Easy to get concentrations wrong. Age of culture etc all seem to effect. Variability going to increase without a doubt. I am generally just doing screening or maybe cloning, so a false negative doesn’t have much lasting consequence.
 

FeelLikeAStranger

How are ya now?
15 Year Member
Direct amplification is great but never consistent. Especially with bacteria. Easy to get concentrations wrong. Age of culture etc all seem to effect. Variability going to increase without a doubt. I am generally just doing screening or maybe cloning, so a false negative doesn’t have much lasting consequence.


Detecting DNA is one thing. I was selling to a forensic lab years ago that was still doing old school Chelex/boil preps for DNA extraction for STR’s/Human Id. Highly fragmented is good for those assays. Same for mouse genotyping. Cut off a toe or chunk of tail and put it with some NaOH, PCR. Lot of genomic DNA in mammalian samples.


Bacteria would defintiely vary, gram + notorious to crack open. Quantitative assays or limited target (highly sensitive) assays are not where you want to use a direct. Qua

viral RNA is another beast. Direct amp is also very dependent on matrix interference. I was trying to find a qPCR mix that was specified for direct amp rt-pcr and that’s tough to find on google. Quanta has one that could work in this regard.

 

Californication

Varsity
10 Year Member
Detecting DNA is one thing. I was selling to a forensic lab years ago that was still doing old school Chelex/boil preps for DNA extraction for STR’s/Human Id. Highly fragmented is good for those assays. Same for mouse genotyping. Cut off a toe or chunk of tail and put it with some NaOH, PCR. Lot of genomic DNA in mammalian samples.


Bacteria would defintiely vary, gram + notorious to crack open. Quantitative assays or limited target (highly sensitive) assays are not where you want to use a direct. Qua

viral RNA is another beast. Direct amp is also very dependent on matrix interference. I was trying to find a qPCR mix that was specified for direct amp rt-pcr and that’s tough to find on google. Quanta has one that could work in this regard.

I did PCR assay development on foods for cyclospora and Hep A while at FDA back in the 90's. I wasn't allowed to work on it very long or very consistently as it was secondary to my real job, but it was a real bitch due to assay interference from the matrix.
 
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FeelLikeAStranger

How are ya now?
15 Year Member
Here’s one of the dangers of relaxing regulations too far...antibody testing is not very accurate, yet. Buyer beware on tests with 15% false positives being used for anything meaningful. At some point, the FDA will need to reign these cowboy non-EUA tests in and pull some off the market.


qPCR tests, while way more sensitive, aren’t as suspect in this process. There are probably some junkier tests out there, but since it is a yes/no decision, it’s really just limit of detection. I havent looked at any of the recent IFU’s for PCR tests and their data , but they’ll probably all fall in that <2% rate for both specificity and sensitivity.
 

FeelLikeAStranger

How are ya now?
15 Year Member

FeelLikeAStranger

How are ya now?
15 Year Member
Here’s one of the dangers of relaxing regulations too far...antibody testing is not very accurate, yet. Buyer beware on tests with 15% false positives being used for anything meaningful. At some point, the FDA will need to reign these cowboy non-EUA tests in and pull some off the market.


qPCR tests, while way more sensitive, aren’t as suspect in this process. There are probably some junkier tests out there, but since it is a yes/no decision, it’s really just limit of detection. I havent looked at any of the recent IFU’s for PCR tests and their data , but they’ll probably all fall in that <2% rate for both specificity and sensitivity.

As Native said, assuming he had the sequence and the proper samples and controls, he could design a qPCR test as good as any out there. The controls and clinical samples are usually the barrier, even with the sequence. You have to be able to clinically validate it. That isnt a problem for anyone right now.
 
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Californication

Varsity
10 Year Member
Native or FLAS, could you discuss how remdesivir works? my understanding it doesn't target RNA polymerase oer se but is a nucleotide analog that disrupts the formation of full length RNA. Does this make it more or less susceptible to becoming ineffective (if it is effective) due to a viral mutation?
 

Native

ToungeInCheek since 2010
Staff member
10 Year Member
Native or FLAS, could you discuss how remdesivir works? my understanding it doesn't target RNA polymerase oer se but is a nucleotide analog that disrupts the formation of full length RNA. Does this make it more or less susceptible to becoming ineffective (if it is effective) due to a viral mutation?


You are correct, that it is a nucleotide analog. So while not truly target the RNA dependent RNA polymerase, the drug acts a on the polymerase. So typically these drugs tent to lack a bit of potency when compared to non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) drugs, due largely to their competitive mode of inhibition and requirement for metabolic activation. (HAART therapy for HIV uses all three classes of drugs in combination).

I don't know for sure, but I would say that viral mutation is less likely. I say this as the nucleotide incorporation domain is still going to have to accept regular nucleotides in order to still work, so the chance of a mutation blocking the analog, while not effecting the other nucleotides becomes less likely. If you look at mutation rates of the virus in general, and check out the latest Covid evolutionary video in my other thread (#41) you see that in general the RNAdepRnaPolymerase is more stable in general as mutation here are selected against, any mutation that effects the fidelity or processivity of the enzyme is less likely.
 

NorthwoodHusker

Junior Varsity
Is there a certain imprinting early on that becomes the prevalent pattern in our immune systems?
And if so, is there to be an expected vatiation of reaction from a vaccine?
 

Native

ToungeInCheek since 2010
Staff member
10 Year Member
Is there a certain imprinting early on that becomes the prevalent pattern in our immune systems?
And if so, is there to be an expected vatiation of reaction from a vaccine?

I might need you to rephrase the question. ;)

Basically you don't lose your ability to diversify antibody repertoire with age.... Sometimes a real young child isn't ready to develop their own yet, and early age isn't a major factor in how you might develop to a vaccine.... I am not sure I am answering the question though.
 
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