# I need a company name who tests for chytrid



## earthfrog (May 18, 2008)

I am doing some shopping around for quotes on qPCR testing for Batrachochytrium dendrobatidis on my quarantined frogs---I am having them tested just for safety's sake before they go into the main tank. Any recommendations?


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## zBrinks (Jul 16, 2006)

Hi Susan,

Drop Aaron (aka frogfarm) a pm - I know he tested his frogs about a year ago, and may be able to direct you to a source.

- Zach


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## earthfrog (May 18, 2008)

Thanks. He recommended Pisces Molecular---I'll look them up on Google.


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## rozdaboff (Feb 27, 2005)

Yes - try Pisces Molecular. The gentleman to contact is John Wood. Their website isn't too flashy - but they are quick to respond to emails or phone calls. If you check the TWI forum - there is also recent post there that gives some more details.

But - Pisces is not running the qPCR assay - they are running a qualitative PCR assay. Not sure if you were specifically looking for the quantitative version or not.


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## pl259 (Feb 27, 2006)

Hey Oz,
What's the difference between a qualitative and quantitative assay? I'd assume qualitative detects if it's there and quantitative gives a measure of how much is there. Is this correct?


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## rozdaboff (Feb 27, 2005)

Exactly. A qualitative PCR assay is a simple yes or no answer. If you get product, then the thing you are looking for was likely in your sample. But - you have no idea how much of it was there.

A quantitative assay on the other hand can tell you (based on a standard) how many copies of what you were looking for were in the sample if it was present. Quantitative assays can be extremely powerful. However - they require more expensive equipment to run and more expensive reagents.

I have only skimmed the surface of the Bd literature - but it appears as if there are two camps - one who believes that the qPCR assay is the gold standard and should be used exclusively, and others that feel that the qualitative assay has its place as well. This is due to the cheaper cost of running the assay and also the source of the sample. Quantitative assays can tell you the number of copies of the thing you are looking for (Bd zoospores for example) - but you need to standardize the size of the sample. If you are doing toe clips - this can be done by weighing the amount of tissue you are extracting DNA from. But - if you are operating from a swab - you can't standardize the amount of sample from swab to swab - so the qPCR assay loses one of its major strengths. And as I mentioned before - it is much more expensive to run - and therefore may limit certain studies.


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## pl259 (Feb 27, 2006)

Thanks Oz! For our purposes, maybe there could be a looser "standard" such that the test indicates presence and if it's over some predetemined threshold. We wouldn't necessarily need to know the exact amounts per mL or whatever. Perhaps as simple as requiring a single swipe per swab, or a 30sec dunk in RO water, or whatever.


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## earthfrog (May 18, 2008)

*Equipment needed for the PCR test*

After you send John an email, he will send you two files---General Instructions for Collecting, Preserving and Shipping PCR B.D. samples, and Methods for obtaining B.D. samples for PCR testing. 

A few notes for clarification to go with these sheets---you can use Everclear from a liquor store as a viable source of ethanol. Some sources are mixed w/petroleum and ruin the specimen, but Everclear is readily accessible. 

They offer a kit of fifty vials, but if you only need a few, consider contacting your local animal hospital for a few vials if you only have a few subjects to test. They will likely hook you up for a nominal fee. The only type of tube that they accept for samples is the 2.0 milliliter, polyethylene (translucent, not clear) screw-capped microcentrifuge tubes with O-ring seals, described here:

http://www.fishersci.com/wps/portal/!ut ... omSearch=Y

Otherwise, you may face a $250 contamination charge if they leak upon opening or are found broken upon arrival.


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## pl259 (Feb 27, 2006)

FYI, The last time I tried buy grain alcohol in New Hampshire, the state liquor store folks told me it wasn't legal here. Don't know how true that is, but it may be true in other states.


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## earthfrog (May 18, 2008)

pl259 said:


> FYI, The last time I tried buy grain alcohol in New Hampshire, the state liquor store folks told me it wasn't legal here. Don't know how true that is, but it may be true in other states.


So, there aren't privately-owned liquor stores in NH? That seems odd if it were true. Everclear is a commercial, non-exclusive product available to anyone of imbibing age.


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## earthfrog (May 18, 2008)

pl259 said:


> FYI, The last time I tried buy grain alcohol in New Hampshire, the state liquor store folks told me it wasn't legal here. Don't know how true that is, but it may be true in other states.


Maybe New Hampshires where they began all that prohibition stuff. You could always drive out to another state to get ethanol/Everclear or order it from Fisher Scientific, the same company above that provides the test tubes. Use histological grade ethanol (Fisher catalog # A962-P4) Do NOT use anhydrous ethanol which contains ethyl acetate, ketones, benzene, gasoline or other organic solvents, which are toxic and will damage the DNA in your sample.


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## heyduke (Sep 19, 2006)

earthfrog said:


> pl259 said:
> 
> 
> > FYI, The last time I tried buy grain alcohol in New Hampshire, the state liquor store folks told me it wasn't legal here. Don't know how true that is, but it may be true in other states.
> ...


some states dictate what kinds of booze can be sold based on alcohol % privately owned or not. ohio is one of those states. only fairly recently (3 or so years) waseverclear made legal here.

sean


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## pl259 (Feb 27, 2006)

As far as I know, all liquor stores in NH are run by the state. There maybe some sort of sub-contracting arrangement available, but I think the state ultimately owns them. However, beer and wine can be bought in just about every grocery/convenience store.

After some quick searching I believe several other states in New England prohibit sales of grain alcohol. At first glance it looks like all three of the states neighboring NH, namely Maine, Vermont, and Massachusetts also prohibit or limit sales. I guess I won't be making any special trips for it. I just asked someone I know in Connecticut to check it out. 

While you can "Live Free or Die" in NH, I guess you can't do it with 190 proof booze!


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## pl259 (Feb 27, 2006)

FWIW,
You can buy grain alcohol at Connecticut liquor stores. Graves is what was available when my friend checked. Interestingly enough Graves is distilled in ME and NH. :roll:


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## Catfur (Oct 5, 2004)

What about plain ol' denatured alcohol from the hardware store? The methanol content shouldn't ruin the sample.


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## zachxbass (Apr 21, 2008)

heyduke said:


> some states dictate what kinds of booze can be sold based on alcohol % privately owned or not. ohio is one of those states. only fairly recently (3 or so years) waseverclear made legal here.


So everclear IS legal here now? i thought it was still illegal (unless you get it on base)

Sorry, kinda hijacked this thread


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## earthfrog (May 18, 2008)

Catfur said:


> What about plain ol' denatured alcohol from the hardware store? The methanol content shouldn't ruin the sample.


John with Pisces Molecular says that it is not as good for perserving the DNA as ethanol---it will increase your odds of getting a false result. I would contact him just to be sure, though, since he justifies the amount of methanol that is in the histological grade ethanol solution---ask him if it's comparable to that.


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