qPCR Detection of Chytrid Fungus
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"I love your website and your qPCR info, I think that many people will find this extremely useful". -- Tiffany Kosch, Ph.D. Candidate, Greenville, North Carolina
This page was developed as part of a free course offered by SAVE THE FROGS! and the Herpetological Circle of Panama. The course, entitled "Instruction and application of quantitative PCR molecular techniques for the study of amphibian epidemics", took place at the Smithsonian Tropical Research Institute in Panama City on October 5th-9th 2009, and was attended by 25 scientists from Panama, Colombia, and Costa Rica. Amphibian population in all three countries have experienced severe declines in numbers due to the chytrid fungus. This course was taught in Spanish by SAVE THE FROGS! Founder & Executive Director Dr. Kerry Kriger, with the valuable assistance of Vicky Flechas of Colombia's Universidad de Los Andes. Thanks to SENACYT for funding the course.
Introduction
Chytridiomycosis is an emerging infectious disease of amphibians that is responsible for mass mortalities, population declines, and species extinctions on several continents. Quantitative (real-time) PCR (hereafter qPCR) is the most reliable means of detecting chytrid infections in both wild and captive amphibians and is the only technique capable of accurately quantifying the number of Batrachochytrium dendrobatidis (causative agent of chytridiomycosis) zoospores present on samples.
While chytridiomycosis has become a major focus of amphibian research worldwide, few batrachologists have any background in qPCR techniques, and training classes are both rare and expensive. The methodologies presented by Boyle et al. (2004) and Kriger et al. (2006b) provide only general descriptions of the qPCR process, and are thus of limited use to those without prior qPCR experience. The ability to perform qPCR in one’s own laboratory would both expedite the processing of samples and decrease the costs incurred by sending samples to private laboratories for diagnosis.
Herein we present step-by-step instructions on using qPCR to detect and quantify the number of Batrachochytrium dendrobatidis zoospores present on skin swabs (Medical Wire & Equipment, MW 100-100; Kriger et al. 2006a). These methods are an in-depth version of those described by Boyle et al. (2004) and Kriger et al. (2006b), and this guide is intended for use by anyone with a modicum of laboratory skills.
This is the most in-depth protocol in existence for the detection of quantitation of Batrachochytrium dendrobatidis. This protocol was developed over countless (often unpaid) hours by SAVE THE FROGS! Executive Director Dr. Kerry Kriger, with invaluable input from Australian qPCR expert Dr. Kevin Ashton. If you find this protocol of use, we strongly encourage you to donate to SAVE THE FROGS! and/or become a member. Your financial support makes pages like this (and qPCR classes) possible...plus, donations to SAVE THE FROGS! are 100% tax-deductible! Please also add a link to this page to your website. If you are a scientist, please consider taking an active role in Save The Frogs Day activities.
Methods Overview
qPCR analysis is performed in three steps:
(1) Extraction of DNA from samples;
(2) Singlicate qPCR assay to detect presence/absence of B. dendrobatidis on samples;
(3) Triplicate qPCR assay of all samples that tested positive in the singlicate qPCR assay, in order to confirm the presence of the fungus and accurately quantify the number of B. dendrobatidis zoospores present.
It should be borne in mind at all times that qPCR is highly sensitive and thus the slightest bit of contamination can compromise results.
View a qPCR slideshow here
The slideshow below is meant to accompany the protocol given below. Please feel free to embed this slideshow on your own website (and add a link to http://savethefrogs.com while you're at it!). You can find the embed code here.
Download the detailed protocol for chytrid qPCR here
This three-part guide, developed by SAVE THE FROGS! Founder & Executive Director Dr. Kerry Kriger, contains:
(1) Detailed instructions on extracting samples; performing qPCR in singlicate; performing qPCR in triplicate (to confirm and quantify the positive samples found during the initial triplicate analysis); using the Applied Biosystems SDS software and ABI 7700 machine (most qPCR systems will be similar, or more user-friendly); and preparing primers, probes and standards that arrive from their manufacturers:
Detailed protocol for the qPCR detection of the chytrid fungus (PDF)
(2) Charts to assist your plate setups for singlicate and triplicate PCR analyses, and for testing standards. (Microsoft Word files)
(3) A detailed list of supplies necessary to perform qPCR, as well as a list of suppliers: Supplies and Suppliers (PDF)
This guide has been downloaded by scientists from Argentina, Australia, Brazil, Canada, Chile, China, Colombia, Dominican Republic, Fiji, Germany, Ghana, Guatemala, Honduras, Spain, Sri Lanka, Thailand, United Kingdom, United States and Uruguay.
Videos from the qPCR course
Coming soon!
Here are some important PDFs you may want to read
Data Analysis PDF from Applied Biosystems
qPCR Essentials from Applied Biosystems
Using Internal Positive Controls from Applied Biosystems
qPCR versus conventional (end-point) PCR from Applied Biosystems
Pipette Upkeep manual by Gilson
How to swab a frog
Here's a one-minute video demonstrating how to swab a frog for chytrid fungus:
The frog in the video above is quite large. If you need to swab a smaller frog, you can hold it as in the photo below (which was taken by fatty acid expert Dave Hall).
Remember to avoid contamination by using a unique plastic bag to catch the frog in, and a non-powdered disposable glove to handle the frog. Be gentle too!
Hey are you a SAVE THE FROGS! member?
You've now downloaded the guide and saved yourself hours or even weeks of frustration, and thousands of dollars of hard-earned grant money. Where to from here? Well now would be a fantastic time to show your support and become a member of SAVE THE FROGS!. Your support makes pages like this possible.
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What they're saying
Dr. Matt Fisher
Imperial College Faculty of Medicine
Department of Infectious Disease Epidemiology
London, United Kingdom
"I like your qPCR protocol: useful and well written".
Dr. Roberto Ibanez
Smithsonian Tropical Research Institute
Panama City, Panama
“This workshop was a vital part of controlling amphibian die-offs in Panama and ensuring that our amphibian rescue efforts pay off. We ran our extractions and they worked perfectly! So, far your protocols are easy to follow, and we are getting good results. It's great!"
Dr. Brian Gratwicke
Panama Amphibian Rescue and Conservation Project
"We have benefitted greatly from your training excercise in Panama and are analysing lots of swabs. Thanks again for instructing that course".
Tiffany Kosch
Ph.D. Candidate
East Carolina University Biology Department -
Greenville, NC
"I love your website and your qPCR info, I think that many people will find it extremely useful".
Dr. Patricia Burrowes
University of Puerto Rico - Department of Biology
I have been reviewing the information on qPCR detection on your website, and I find it very helpful –this is a great contribution, thank you!
Dr. Tobias Eisenberg
Veterinarian - Langgöns Germany
"Great job!
Thanks a lot for publishing all this information here!
We are running chytrid qPCR in Germany as the only lab.
Would be great if you could keep us informed!
Take care,
Tobias"
Steven Whitfield
Ph.D. Candidate
Florida International University - Miami, FL
"Good job on the qPCR course in Panama. Capacity building is where it's at!"
Dr. John Cossel
Chair, Department of Biology -
School of Health and Science
Northwest Nazarene University - Nampa, ID
"I want to thank you for the great website you have made available to the public and researchers alike! Thank you as well for the posted protocol! We just purchased our own qPCR machine and hope to do our own analyses in the future using your posted materials."
Dr. Dede Olson
Research Ecologist
- USDA Forest Service
Corvallis, Oregon
"Excellent!!!!"
Alejandra Zamora
Guatemala
"Your web site is incredible! The PCR protocol really helped me."
Lilly Eluvathingal
Ph.D. Candidate
Florida International University -
Miami, FL
"Good job....It's so cool to see people working together for an important cause".
Here's what students said about the qPCR course
“Magnifico que realicer estas actividades. Espero que se forme un buen grupo de analysis de quitrido y que sigan obteniendo apoyo.”
“El curso fue extraordinario, seria bueno se repitieran.“
“Creo que ha sido el curso mas complete dictado en Centro y Suramerica sobre esta tecnica. Lleno completemente mis expectativas.”
Website design, photos & content by Kerry Kriger unless otherwise noted.
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qPCR Chytrid Fungus Protocol