Suggestions for viral diagnostics

[Rick Byers]

Hi,
I'm curious to better understand the pattern of viral infections in my
family (I've got two young kids, so it's pretty common for some cold
to be going around our house). I'm trying to figure out the most
practical way to do regular URT diagnostics. I've got no lab
experience (but would love to learn) and almost no lab supplies (but
am willing to invest up to a few thousand dollars as necessary).
Ideally I'd like something that is both quantitative (so I can track
viral load over the course of infection) and multiplexed (so I can
identify which viruses are responsible and catch most common ones).

I know I can do some simple diagnostics at home with a modest
investment. Eg., I considered doing simple plaque assays or simple
PCR runs, but I'm hoping I can find something that's less time
consuming at non-trivial scale (eg. detecting at least 10 different
viruses/strains, ideally many samples over a period of time). Perhaps
there's an immunoassay panel of some sort I could use at home? Or
perhaps the best option is a service that will do a multiplexed qPCR
run for me on a set of samples I send in.

I'm continuing to do research of various products and diagnostics
services, but I'm finding it pretty difficult to get specific prices
and weigh the options. Any advice would be greatly appreciated.
Thanks!

[Cathal Garvey]

Hey Rick,
I think PCR is almost certainly the way to go, for a few reasons.

Firstly, you're talking about mammalian viruses; to grow them (which is
a bad idea in any case), you'd need mammalian cells, and mammalian cells
call for more intensive care than bacteria. It's a big investment, and
it requires a lot of time and effort just to keep them going.

And that's ignoring the fact that each virus will call for a different
target cell type, which in turn calls for different care procedures;
kidney cells may need very different medium and care than liver cells,
for example.

Secondly, plaque assays don't really work, AFAIK, for mammalian viruses.
Because they must be grown with a liquid layer on top, and that liquid
layer allows viruses to spread freely, instead of growing outwards like
bacteriophages.

Besides, if dealing with human infections, a good rule #1 is *never
culture*. If you can do your work without culturing the viruses at all,
then you should.

DNA survives boiling and alcohol, but viruses don't. If you want to
study your kids' colds, you could just take a sample of nasal mucous,
boil it in an eppendorf tube for 10 minutes, add 40% EtOH just to be
sure and then use a tiny sample for PCR, using primers that will amplify
your virus of interest. Ideally use primers that are already tested and
shown to work reliably in the available science literature. If you make
your own, aim for 25 nucleotides in length in a non-repetitive area of
the virus genome that's probably well-conserved, such as promoters.

PCR is great for many reasons. One reason is that it allows you to study
something that is impossible or unwise to culture, such as thermophiles,
bizzarre soil symbiotes, or human pathogens. I would strongly suggest
sticking with PCR and taking normal precautions to avoid infecting
yourself while you take samples.

--
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PGP Public Key: http://bit.ly/CathalGKey

[Rick Byers]

Thank you very much Cathal! This is great - matches up with what I've
found in my research (I really didn't want to have to culture, and PCR
is more sensitive anyway). Thanks in particular for the tips on
boiling and mixing with alcohol.

Now, are you suggesting I just run gels at home? This is certainly an
option, but I get the feeling this will be pretty labor (and reagent)
intensive compared to relying on a lab equipped with the right
automation (especially because for each sample I might need to test it
against >10 different viruses). Also I'd love to get some indication
of viral load if possible. Multiplexed qPCR seems to be an emerging
standard here, but I'm not sure how to find a lab I can buy services
from on samples I send (and, at first glance at least, a qPCR machine
is probably still too expensive for me to buy myself). As an example,
here's a couple of the promising products I've found for multiplexed
qPCR respiratory virus panels:
http://www.luminexcorp.com/Products/Assays/ClinicalDiagnostics/xTAGRVP/index.htm
http://www.idahotech.com/FilmArray/RespiratoryTest.html

Another option (if I give up on measuring viral load) is Sanger
sequencing - at least there are lots of cheap services advertised
here. Perhaps with carefully chosen primers I could identify the most
common respiratory viruses with ~10 reactions (which looks like I
should be able to get for <$50/sample). Getting a bit of sequence
would be a nice bonus for eventually tracking specific viral strains.
Is this a viable option worth considering do you think?

Thanks!
Rick

[Chris]

I am very interested in what you are trying to do Rick. Seems like a
concept that would mate up very well with "Tricorder X Prize"

http://www.qualcommtricorderxprize.org/

Sounds like your timescale for a device / service is a little shorter
then 5 years of development though.

I assume you are ultimately interested in understanding the 'bugs'
that are making their way through your house so you can better treat
everyone or is it more of curiosity at this point? Any chance you
could mate up with a local university or small company that could be
the point of sale for purchasing reagents / sending off services. I
understand your frustration with trying to get prices on products.
When I try and purchase something with my personal email account /
home address I usually get no where. I usually use my work email /
work address (I own a small business) and its night and day how I get
better service and prices quite quickly.

Chris

On Jan 31, 8:41 pm, Rick Byers <r...@rbyers.net> wrote:
> Thank you very much Cathal!  This is great - matches up with what I've
> found in my research (I really didn't want to have to culture, and PCR
> is more sensitive anyway).  Thanks in particular for the tips on
> boiling and mixing with alcohol.
>
> Now, are you suggesting I just run gels at home?  This is certainly an
> option, but I get the feeling this will be pretty labor (and reagent)
> intensive compared to relying on a lab equipped with the right
> automation (especially because for each sample I might need to test it
> against >10 different viruses).  Also I'd love to get some indication
> of viral load if possible.  Multiplexed qPCR seems to be an emerging
> standard here, but I'm not sure how to find a lab I can buy services
> from on samples I send (and, at first glance at least, a qPCR machine
> is probably still too expensive for me to buy myself).  As an example,
> here's a couple of the promising products I've found for multiplexed

> qPCR respiratory virus panels:http://www.luminexcorp.com/Products/Assays/ClinicalDiagnostics/xTAGRV...http://www.idahotech.com/FilmArray/RespiratoryTest.html

[Rick Byers]

Thanks for the suggestions Chris!

I'd love to believe that better understanding the bugs moving through my house would ultimately lead to better treatments and prevention.  Big picture, with sequencing costs falling so rapidly, I can imagine a 23AndMe/Facebook around our microbiome - using regular deep sequencing to track infection patterns and provide insights that would change people's behavior (privacy issues aside, I see no technical reason why I can't know with reasonably certainty who exactly I contracted a bug from - or more importantly who exactly I infected by going to work sick, etc.).  That said, having little formal training or experience in biology (I'm a software engineer at Google), I'm trying to be practical and focus for now just on satisfying my curiosity to better understand the patterns of infection I'm involved in.  I've got a list of a few concrete questions I'd like to answer and other brainstorming at http://goo.gl/9h7uI, I'd love any feedback anyone has (should be able to put comments directly in the doc if you like).

The Qualcomm X Prize sounds great - thanks for the link!  I've followed to many other X Prizes, I can't believe I hadn't heard of that one before.

Thanks for the tips on ordering.  I've started to research the microbiology department at the local University (Waterloo, Canada).  I was thinking I should try to get a bit further myself (or maybe sign up for a class) before I'm likely to get anyone there interested in helping me.  I took some genome sciences classes at U of Washington a couple years ago when I lived in Seattle - I'm wishing now I started this work while I still lived there <grin>.

Thanks,

   Rick

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