06-16-2008, 05:03 PM
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Join Date: Nov 2004
Location: New England
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Re: Some questions about chytrid
So I emailed to see about the 'proceedure' and indeed they responded rapidly [2hours].
The response I am posting here b/c I think it has some good info for all those interested.
Dear Shawn
We would be pleased to PCR test amphibian samples for Batrachochytrium dendrobaditis for you; we test samples for many private individuals.
Attached are two files which answer most of the common questions about collecting and preserving samples for B.d. PCR testing. The first is a protocol for collecting amphibian toe clip, skin scrape or skin swab samples for PCR testing from Lauren Livo whom we work with at the Colorado Division of Wildlife. The second file from Pisces continues on from Lauren’s with instructions and information about packaging and sending samples for B.d PCR to us.
Several additional notes beyond what is covered in these two files:
Because our experience with the 2 ml screw-capped microfuge tubes with a captive O-ring gasket recommended in the instruction files has been very positive - nearly zero leaking tubes in thousands of samples - we strongly recommend using these tubes for your samples. In response to requests from many customers who don't want to buy 500 vials at a time, or don't want to spend the time to be sure they're getting the right tubes or ethanol, we are now offering "swab sample collection kits" consisting of 50 2ml screw-cap tubes filled with 1 ml of 70% ethanol; 50 swabs; ziploc bags for everything; and a box that can be used for return shipping to Pisces. The cost of the 50-sample kit is $50 plus shipping.
The "general instruction ..." file specifies the materials for pooling samples, but doesn't yet explain the two alternative sample pooling strategies we have developed and offer. We can pool up to 8 swab samples into one DNA prep+PCR sample. Either you can do the pooling, or we can do it here. Although there is an extra charge if we do the pooling (see below), we generally recommend that new customers let us do it, at least for the first pass of sampling at new sites where nothing is known about the prevalence or severity of B.d. The chytrid PCR assay can detect less than 1 zoospore, so it is extremely sensitive to sample cross-contamination; we have procedures in place to pool samples efficiently without cross-contamination. Also, if we do the pooling, we can pool 1/2 (by volume) of each individual sample. Then if a given pooled sample is PCR positive, we can go back and prep and PCR test the remaining half samples individually. This works out well when one wants to know a true B.d. incidence level without spending lots of money on a large batch of samples that all turn out to be PCR negative.
We do not yet require or have specific sample submission forms; all we require are: 1) The name of the sample submitter; 2) A hard copy list of the number of samples submitted and the individual sample IDs; and 3) To whom the report and the invoice should be sent.
The current price for carrying out the DNA extraction and the chytrid PCR test for samples sent to us is US$27 per sample. For samples that you send as individual samples and we do the pooling, there is additional $35 pooling charge for each group pooled (for example, 8 individual samples pooled into one DNA prep + one PCR test would cost:$35+$27 = $62. For samples sent to us already pooled, the charge is $37/pooled sample. Samples sent in tubes or vials other than those recommended are subject to a 20% surcharge. Samples which leak in transit – and therefore require extensive additional anti-cross contamination measures at Pisces - will be subject to a $250 per batch leak surcharge. If we discover a leak in a sample batch, we will immediately email or call you to report the leak and allow you to decide whether we should proceed, or let you resample and submit another (hopefully) non-leaky batch of samples. Individual swab sample collection kits are $50 per 50 samples, plus shipping. Kits for customer pooled samples (50 ml tubes with 70% ethanol and 8 swabs per tube, instructions) are $80 for 10 pooled samples.
Our terms are: Payment is due upon receipt of the sample test results report. All we require from new customers is a name and address to send the invoice to, and a P.O. number if required by the customer. Unfortunately, we are not able to accept credit card payments at this time. Samples should be packed per the instructions in the "General instructions" and shipped FedEx, DHL or UPS; 2nd day delivery is fine.
Our normal turn-around time from receipt is two to four weeks. However during the summer field season, or in the fall when all the field season samples have been sent in, our turn-around can slow to four to six weeks. If you have a need for faster results, for animals in quarantine or a report deadline, please call us. Although we haven’t completely formalized our rush sample times and charges, we can very likely work something out that meets your needs – our fastest sample turnaround time to date was 9 hours (FedEx Priority delivery of the samples in the morning; PCR results emailed by 6 PM!) (currently we have been quoting < 1 week turnaround for $54/sample and < 48 hr turnaround for $108/sample). We will email and/or send out a signed hard copy of the test results report, whichever is needed by the sample submitter. Test results are kept confidential and not shared with anyone other than the sample submitter or their organization without express permission from the submitter.
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Shawn Harrington, Moderator Staff
TWI/ASN; DB Dedicated Supporter
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