eDNA - Single-species assays (qPCR)

Single-species assays using quantitative PCR (qPCR) approaches have become powerful tools for detecting and monitoring individual species in environmental DNA samples. By targeting species-specific genetic markers, qPCR assays can sensitively detect and quantify trace amounts of this DNA from environmental samples such as water, soil, or sediment—without requiring the physical presence of the organism. 

For all environmental DNA-based applications, it is important to have a basic understanding of what a PCR (polymerase chain reaction) is. PCR is a laboratory technique that amplifies, or makes many copies of, specific segments of genetic material from a sample. It is a highly sensitive and accurate method used to detect tiny amounts of genetic material. These targeted segments can be specific to a particular species (e.g. Chinook salmon) or group of organisms (e.g. Fish).

When to use this approach

The integration of qPCR with eDNA sampling allows for high specificity and sensitivity in detecting target species, making it especially valuable for monitoring rare, invasive, endangered, or cryptic species. Broadly, this approach is used when you are interested in quantifying the amount of DNA present from a single species, or from a select few species, in an environmental DNA sample. 

For example, you would not use this approach if you are interested understanding the composition of the entire fish community at a specific location but you would use it if you are specifically interested in determining how much DNA from a specific fish, eulachon for example, was present at a particular location and whether there was more eulachon DNA at site A versus site B. In the case of an economically and/or culturally important species, this information could help you better manage your fishery.

You might also consider using single-species assays if you are trying to track the emergence of an invasive species that you know to be approaching your region. In this case, the amount of DNA from the invasive species will be a very small component of the total environmental DNA pool when it has yet to become established and may not be easily detected using whole-community based approaches (it is hard to find a need in a haystack, after all). A specific-specific assay approach will allow you to ‘look for’ only the DNA from that invasive species, while ignoring the rest of the eDNA pool (think of it as the metal detector for that needle). The quantitative nature of this approach also allows you to count the DNA copies in a sample and understand when and where an invasive species is taking hold; information that can guide active management and mitigation of an invasion, ideally before it becomes too established.

Input

The starting material for single-species qPCR assays is a standard environmental DNA sample that can be collected from water, soil, sediment, air, etc. 

Additionally, you will need to have, or develop, a genetic assay that targets your species of interest. The iTrackDNA project team has worked hard to develop and/or validate qPCR assays for many species of interest in Canada and you can find the list here. 

The assay components are then mixed together in a specific recipe and run or analyzed on a quantitative PCR (qPCR) or droplet digital PCR (ddPCR) machine. While there are differences between these two technologies, the ultimate output will be similar. Typically, samples are run in replicates, between 3 and 8 per sample, to estimate the prevalence of detection of your target, in addition to quantifying the amount of target DNA in your sample. 

Output

Most simply, the output of single-species assays includes the sample ID and the number of gene copies estimated per sample (e.g. per litre of water, per mg of sediment, etc.). However, there are calculations using the raw outputs from qPCR or ddPCR machines that must be made to estimate copies per sample. 

For example, in the case of qPCR a standard curve of synthetic target DNA is run alongside the samples. From this, the copies of target DNA per qPCR reaction can be estimated for each sample and then converted to copies of target DNA per sample. ddPCR technology does not require the use of a standard curve when running samples as it generates an absolute quantification of droplets containing the target DNA, from which the total number of copies in a reaction is estimated. Even still, a few synthetic standards are commonly run alongside samples being run using ddPCR technology to constrain between-run variation. In each case, copies per PCR reaction is estimated and then converted to copies per unit of sample (e.g. litre) using information like the volume of water filtered, the volume of DNA extracted, and the volume of DNA used in the PCR reaction.

Pros

Single-species assays provide quantitative information about your organism of interest. While they do not yield actual counts of the organism (number of individuals) as you might get from a visual survey, they do give an idea of the amount of DNA of that target species present in the environment which should be more in locations where more of that species is present and less in locations where the species is rare or absent. 

Cons

Since one must make a choice about species to focus on for single-species assays, you do lose the ability to understand the total community composition and the relative abundance of your focal organism in the whole community. While the cost for this assay may be cheaper than for metabarcoding, you only gain information about one organism so the cost per organism is ultimately more expensive in single-species assays compared to community-based assays.