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
On Jan 31, 10:35 am, Cathal Garvey <cathalgar...@gmail.com> wrote:
> 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.
>
> On 25/01/12 05:51, Rick Byers wrote:
>
>
>
>
>
>
>
>
>
> > 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!
>
> --www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key:http://bit.ly/CathalGKey
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