Zika Virus Detection

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Bryan Daniels

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Mar 28, 2016, 5:53:34 AM3/28/16
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I am just back from Spring Break in Puerto Rico and have come down with a 103 F fever, sore eyes etc. CDC has classified PR as high risk Zika zone. Most likely I have influenza, but on the off-chance it is Zika, I thought it might be an interesting DIY bio project. (I am male and past pregnancy stages in our family. . .)

I have poked around on-line and there appear to be PCR based diagnostic tests, so there are clearly primers available. Also found this article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285245/

I'm wondering if anyone in DIY bio community has tried this test themselves (on the cheap). As I'm not feeling too well right now, so I am also interested in how to preserve samples (and what type of samples) for later use. Any straight-forward protocols, would be much appreciated.

I have a reasonably well equipped basic lab, but only ordinary PCR capabilities, I.e., no real-time OCR.

Thanks in advance.

Jake

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Mar 30, 2016, 8:06:19 PM3/30/16
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Haven't tried this as I'm not in a Zika zone or traveled through one.  I would think that as long as you have a thermocycler or are willing to do it manually... It should be simple enough.
Extract DNA, add primers and master mix, cycle, dye, and spec or run on gel.
I might be interested in trying this if I can get a presumed positive sample...  You did draw a vial of blood and freeze it right?

-Jake


Dakota Hamill

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Mar 30, 2016, 8:18:15 PM3/30/16
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I'm all for affordable and accessible diagnostics, but you should probably go to the doctors and not send potentially hazardous infectious self-drawn blood samples to a stranger from the internet.

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Jake

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Mar 30, 2016, 8:51:57 PM3/30/16
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I was under the impression that this was a matter of curiosity rather than a medical issue...  but yes, it is always better to outsource your medical needs to non-internet strangers, or internet based strangers that have paid their dues to the powers that be.

I would point out though that there's nothing wrong about drawing your own samples, every sample is mailed to a lab somewhere, and all blood products are considered potentially hazardous regardless of the source.

Dakota Hamill

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Mar 30, 2016, 9:20:31 PM3/30/16
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Let me preface my point with a story.

About 5 years ago or so I went to a Genome conference in Boston.  In between talks I was in the lobby and mingling with people, some around my own age at the time, early twenties.  I met a kid and we got to talking.  I said I was involved at a local community lab in Boston and did research stuff at home or at school on the side.  He asked what we did at the lab and I said we did GFP transformations, DNA barcoding, etc.  Basic molecular biology.

He said that was neat and all, but what he really wanted to do, and I'm not making this up was -- "I want to make like, a crazy virus, and put it on random door-handles"

I'll never forget that.

All of us in this community have a responsibility to ourselves, the world, the public, and each-other, to be exemplars of responsible citizen scientists.  There's a person on this group that waited years for a license just to be able to do a GFP transformation outside of a lab in an EU country.

We enjoy a lot more freedom in the US for at-home or extra curricular science and engineering.  No doubt though, garage and basement scientists are under scrutiny, whether it be from some invisible all-seeing government entity, the media, the general public, or even each-other.

I'm not saying don't go off the beaten path and be creative, inventive, or resourceful.  I applaud your enthusiasm and tenacity, get that shit done.

All I'm saying is each and every one of us could be in the spotlight, and we should maintain a certain level of care in what we do, for our own well being, our neighbors well being, and the community we're all a part of here.

I don't know your background, skills, your circumstances, or access to space.   You could be ultra-qualified to be processing blood samples and could be working in a university lab space you rent or are a part of where it's safe to handle these type of specimens.   

I'm not trying to put you on blast here, I'm just saying, would you want someone you don't know processing human blood contaminated with Zika virus in a salad spinner centrifuge and lightbulb PCR machine in the apartment next door, and throwing everything out in the trash?


Jake

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Mar 30, 2016, 10:00:15 PM3/30/16
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Quick note to amend my random comments earlier... Blood is always potentially hazardous.  I'm not volunteering to do anything with blood, just a poorly worded comment.

What I wanted to suggest is that he might take a sample, freeze it, and use his previously mentioned PCR capability to check for amplification.  If I were in his situation that's what I'd do out of curiosity.  It shouldn't be terribly difficult or expensive to do and would make an interesting project.

djwr...@gmail.com

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Mar 30, 2016, 11:43:23 PM3/30/16
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Identifying which particular disease you have is valuable information that many people even in developed countries have difficulty obtaining.

For instance,  I had the flu but have no idea which strain. My decision of whether or not to get a flue shot next year would change depending on if the one I had, and am now immune is included.  Rapid diagnosis in bacterial meningitis is crucial because it can kill so quickly and PCR  is fast. IIRC Eva Harris's book "PCR at Home" brought a then new technology to Central America to rapidly identify  dengue fever. This was, of course, used by by medical professionals. 

"Currently, the standard assay for Zika viral infection is a PCR test that probes for the presence of viral RNA in a sample. While it works well to detect the virus, the pathogen’s RNA is only around for a short period of time. “By the time [patients] make it into the clinic, the virus is likely gone or it’s at the tail end, beyond the limit of detection,” said Vasilakis."

A urine test is better than blood according to this paper.
Detection of Zika Virus in Urine

I think many US hospitals underutilize  PCR as a diagnostic tool. If you can safely come up with a protocol to do it yourself it is a worthwhile endeavor that would lead to wider implementation of PCR diagnostics.  

Jake

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Mar 31, 2016, 3:23:41 PM3/31/16
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Found a primer sequence here...
(Table 3)

Forward primer = AARTACACATACCARAACAAAGTGGT
Reverse primer = TCCRCTCCCYCTYTGGTCTTG

Here's a good site for aligning virus sequence...

So you'd need an extraction kit like QIAamp Viral RNA Mini Kit, a one-step RT-PCR kit, the primers, and a gel or spec for measuring amplification.  A bit expensive for a one-off test, but you can probably get samples if you shop around.
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