Limits of detection and limits of quantification for Longfin Smelt (Hooligans/Hoolies)
Based on the reading: Reporting the limits of detection and quantification for environmental DNA assays Klymus et al., 2019
Environmental DNA (eDNA) is used to find rare species and is being used in fishery and wildlife conservation and management strategies (for a definition of eDNA see the previous blog post). There is a need for standardization of eDNA methods and reporting. Limit of detection (LOD) and limit of quantification (LOQ) are not fully established for eDNA studies. At the SSRC we use quantitative PCR (qPCR) to detect eDNA of Longfin Smelt (a.k.a. Hooligans (a.k.a. Hoolies)) to detect low concentrations of eDNA in the Nooksack River and Bellingham Bay.
However, we not only want to know of the Hoolies are present, we also want to quantify them. There is confusion about how to define LOD and LOQ for qPCR studies. Conventional definitions of LOD and LOQ do not fit qPCR data - because the previous definitions require linear response. Data from qPCR are not linear and negative samples do not produce signal distinguishable from background signal. Therefore, Klymus and others (2019) sought to refine and standardize LOD and LOQ for use with qPCR.
"Our goal is to help establish standard LOD and LOQ definitions and guidelines to improve communication, inform data interpretation, and facilitate cross‐study comparisons, all of which support conservation and management decisions." Klymus et al., 2019
LOD in qPCR can be defined as the lowest concentration of target analyte that can be detected with a defined level of confidence.
"The LOD is based on detection/nondetection criteria and describes an assay's ability to detect the target sequence at low levels." Klymus et al., 2019
LOQ is the lowest amount of analyte in a sample that can be quantitatively determined with stated experimental conditions.
"...measurement of concentration is addressed by the LOQ, which reflects the assay's capacity to precisely quantify copy number." Klymus et al., 2019
For qPCR, precision can be assessed using the coefficient of variations (CV) of the measured concentrations of standards. Data from the replicate standard curves are evaluated as the
binary, qualitative outcome for LOD (detection/nondetection) and as the CV for LOQ:
Klymus and others (2019) brought together data from seven different laboratories, each used synthetic DNA (except on group used plasmid) for their standard curves.
The SSRC is using synthetic DNA. We need to be certain of the amount of target molecules present in test reactions. With synthetic DNA, a known concentration of DNA can be tested. We recently designed a segment of DNA that will be used as our standard for Hoolies.
Side note: in a dream I had last night, I envisioned the synthetic DNA of the Hoolies as a hologram that appeared between my palms.
To determine LOD and LOQ for an assay, standard concentrations of DNA must be used in many qPCR experiments. For LOD, they must also include a mix of positive and negative replicates. The accuracy and precision of LOD and LOQ calculations increase with replication.
Klymus and others (2019) suggest either (1) discrete thresholds for determining the LOD and LOQ for an eDNA qPCR assay, or (2) calculations to determine LOD and LOQ based on curve fitting performed by an R script (drc).
To facilitate the evaluation of eDNA assays Klymus and others suggest, at a minimum, along with parameters by Bustin et al., 2009 and Goldberg et al., 2016, do the following:
Reported LOD and LOQ values should be accompanied by:
the concentration range and number of replicate standards per concentration used for calculating LOD and LOQ.
the determination approach used (either the discrete threshold or curve-fitting modeling method) and,
the specific criteria for LOD probability of detection and LOQ precision that were applied.