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Detecting pathogens

Field work is only one side of the work done in P³. Once all the different samples have been collected and transferred to all the different freezers and fridges of the P³ partners the demanding lab work is starting. Surely, extracting e.g. skin swabs from amphibians, is less glamorous as hiking through the mountains and catching amphibian tadpoles, it is crucial to determine how many amphibians are infected with the different pathogens.

So, how does this work? Hundreds of plastic tubes containing long cotton swabs are brought into the lab. They have come from the field and each has a skin surface sample from one individual. The first step is to extract the DNA from these samples. The tip of the cotton swab is combined with glass beads and an extraction buffer in a small plastic tube. They are shaken together in a homogenizer so the beads break the cells and expose the DNA. Centrifugation and heating separates the DNA from the junk and the DNA is transferred to a clean tube.

Now that we have pure DNA, it’s time to see if there is any pathogen DNA in the samples. The samples are prepared for the qPCR machine, which amplifies tiny amounts of DNA by repeated amplification steps. The technique is so sensitive that even if only one spore of Chytrid is present, we’ll still be able to detect it. Primers specific to the pathogen of interest are used so that we only see the amplification of this particular DNA. As an end result, the screen on the qPCR machine shows how the DNA was amplified and we can determine exactly which samples contain the fungus and how much of it (the infection load).

With the proportion of infected individuals (prevalence) and their infection load we then can analyse, which environmental factors are playing a role in infecting amphibians, a major axis of the P³ project.

#Pyrenees #pathogens #Amphibianchytrid #labwork

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