The molecular diagnostics facility at the Sierra Nevada Aquatic Research Laboratory conducts testing of samples using quantitative PCR (qPCR) assays. We provide an at-cost service to detect the amphibian chytrid fungus (Batrachochytrium dendrobatidis – Bd) from skin swab samples.
If you are interested in using this service, please fill out the qPCR Request Form by clicking on the button below. If you have additional questions, contact Alexa Lindauer (Project and Laboratory Manager, alindauer[at]ucsb.edu). Please read our protocol for collecting and analyzing amphibian skin swabs for Bd prior to submitting a qPCR Request Form to ensure that a) your samples can be processed in our lab and b) our protocols and results will be sufficient for your project. Information on qPCR analysis, results interpretation, pricing, timeline, and sample collection are included below.
qPCR analysis and results interpretation
We analyze skin swab samples using a Prepman extraction and standard qPCR methods as described in Boyle et al. (2004). Unless otherwise requested, samples will be run singly instead of in triplicate. This Bd qPCR assay targets a portion of the ITS1 gene for amplification. Because we use an ITS1 plasmid standard rather than a standard made from a known quantity of zoospores, results are expressed as the number of copies of the ITS1 gene per swab. For more information on qPCR protocol and how to interpret your results, please read our Collection and Analysis of Amphibian Skin Swabs for qPCR Analysis of Bd Load protocol.
Current pricing is $8.00/sample for clients associated with the University of California, and $12.68/sample for non-UC clients. We are dedicated to making amphibian disease testing more accessible and therefore do not profit from Bd qPCR services. Sample cost covers consumables and reagents, equipment maintenance, technician time, and administrative costs required by the UC system (the difference in pricing between UC- and non-UC clients covers the required overhead cost the UC system charges to process external payments). After we process your samples and share your results, you will receive an invoice which can be paid with a credit card.
We are happy to provide an at-cost service to clients and collaborators to encourage disease sampling in amphibian populations. However, because molecular diagnostics are a small part of our research program, we cannot always promise a fast turn-around from sample shipment to processing. Please keep in mind that April through September is our busy season with the highest volume of samples and fewest work hours dedicated to the lab while we prioritize work on internal research projects and field work.
In addition, due to COVID-19, turnaround can be slowed further by supply chain issues and backlogs on qPCR consumables and reagents. If you have a large number of samples (>150), please submit a qPCR Request Form ASAP so we can order enough consumables and reagents to process your samples. Supply chain backlogs may delay sample processing by 3-6 months.
Organize your samples in numerical order in cardboard or plastic freezer boxes. If you cannot obtain freezer boxes (due to budgetary restrictions), please organize samples in numerical order in groups of no more than 20 in Ziploc bags labeled with the swab ID range contained within. Place your samples in a cooler with water ice (dry ice is not necessary) and ship samples to:
Sierra Nevada Aquatic Research Laboratory
Alexa Lindauer, Molecular Laboratory
1016 Mount Morrison Road
Mammoth Lakes, CA 93546
– Choose overnight shipping with FedEx or UPS (never USPS).
– Ship on a Monday, Tuesday, or Wednesday to avoid shipping delays that could keep your samples warming in a warehouse over the weekend.
– DO NOT ship unless you have confirmed with Alexa Lindauer that she can receive samples. Send Alexa the tracking number after shipping. You may also request an in-person drop-off if convenient.
Sample collection guidelines
Samples must be swabs that were a) completely air-dried upon collection or b) were placed on ice or in a refrigerator or freezer immediately or shortly (within a few hours) after collection. Damp or wet samples that have not been kept cool are likely to have bacterial or fungal growth that may compromise sample integrity. We do not accept samples in ethanol. We use Medwire MW113 swabs.
Swab samples must be contained in 1.5mL microfuge tubes that meet the following specifications (ex: Fisher 02-707-353) :
– round bottom shape
– tethered screw cap closure
– suitable for boiling
– withstand 10,621 rcf
– DNase and RNase free
– sterile (preferable, although at the discretion of the researcher)
– labeled with pre-printed cryo-label
A round bottom is necessary for the DNA extraction process due to the configuration of the heat plates we use. A tethered cap improves efficiency in the DNA extraction process and reduces the chance of sample mix-up (i.e. placing the wrong cap on the wrong vial during DNA extraction). All vials must be labeled with a swab ID, printed on a cryo-label and adhered to the top of the cap. Swab IDs must be unique both within and among all research groups and qPCR clients. Please contact Alexa Lindauer (firstname.lastname@example.org) before printing swab ID labels to ensure the use of unique IDs for every sample you submit. If you are purchasing your own vial labels, be sure to buy labels with adhesive that can withstand -80 °C, the temperature at which your processed DNA extracts will be stored (ex: 3/8″ Tough-Spots cryolabel sheets).