The fungus that causes chytridiomycosis in amphibians develops in their keratinized cells, so we sampled adult skin and the oral cavity of the larvae. To do this, we only need to rub these areas with sterile cotton, which causes no harm to the animal.
Once in the laboratory, we extract the DNA from the sample using specific reagents and a series of agitating treatments, centrifugation and heat. This process breaks down the cells releasing their contents, and then we purify this by removing most elements that are not DNA. Thus, we have isolated the genetic material present in the sample, which will be a mixture of amphibian DNA and of the many bacteria from their skin and, if infected, the chytrid fungus itself.
To detect the DNA of the fungal pathogen, we carry out a process known as polymerase chain reaction (or PCR). In particular, we use a variant of this technique: quantitative PCR or real-time PCR, which not only allows us to detect DNA that we seek, but the exact amount present in the sample, which indicates the degree of infection in the amphibians studied.
This PCR process is carried out in a machine known as Thermal Cycler, using plates which can analyse up to 43 samples in duplicate. This device performs successive cycles at different temperatures necessary for the reaction to occur and after two hours of waiting, we get the results.
This technique is complex and also quite expensive. To analyse each sample will cost around 5 € in materials and to adequately know the status of a population of amphibians (e.g., a source tadpoles), we need at least 20 samples. In addition to the cost of materials, to process 20 samples takes a full day of work in the laboratory. As you can imagine, to analyse the infection status of an area requires an analysis of dozens of amphibian populations of each species. On the other hand, when performing controlled field or laboratory experiments, the number of samples needed makes the costs soar to thousands of euros. This is one of the main causes why research into emerging diseases is so costly and slow.
Thanks to the BBVA Foundation for covering much of the laboratory costs of this project!