Conventional versus real‐time quantitative PCR for rare species detection

Abstract

Detection of species in nature at very low abundance requires innovative methods. Conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) are two widely used approaches employed in environmental DNA (eDNA) detection, though lack of a comprehensive comparison of them impedes method selection. Here we test detection capacity and false negative rate of both approaches using samples with different expected complexities. We compared cPCR and qPCR to detect invasive, biofouling golden mussels (Limnoperna fortunei), in samples from laboratory aquaria and irrigation channels where this mussel was known to occur in central China. Where applicable, the limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate of each PCR method were tested. Quantitative PCR achieved a lower LoD than cPCR (1 × 10−7 vs. 10−6 ng/μl) and had a higher detection rate for both laboratory (100% vs. 87.9%) and field (68.6% vs. 47.1%) samples. Field water samples could only be quantified at a higher concentration than laboratory aquaria and total genomic DNA, indicating inhibition with environmental samples. The false negative rate was inversely related to the number of sample replicates. Target eDNA concentration was negatively related to distance from sampling sites to the water (and animal) source. Detection capacity difference between cPCR and qPCR for genomic DNA and laboratory aquaria can be translated to field water samples, and the latter should be prioritized in rare species detection. Field environmental samples may involve more complexities—such as inhibitors—than laboratory aquaria samples, requiring more target DNA. Extensive sampling is critical in field applications using either approach to reduce false negatives.

Detecting a spreading non-indigenous species using multiple methodologies

“…FlowCam has the advantage that it can process large numbers of samples and present candidate images to the operator for confirmation with significantly lower operator fatigue than CPLM, at a cost of lower sensitivity and relatively high startup costs. If a highly sensitive eDNA approach were desired, the researcher could use qPCR (at higher startup and running cost than cPCR), as qPCR is up to 10Â more sensitive than cPCR (1 Â 10 À7 vs. 1 Â 10 À6 ng/mL; Xia et al 2018), making detection more likely.…”

“…Total genomic DNA was extracted from a 1 mL subsample of each filtered bulk plankton sample (the same samples as previously used for CPLM and FlowCam), representing between 0.02 and 0.06 m 3 of lake water sampled (Zaiko et al 2015, Ardura et al 2017. We based our choice of conventional PCR (instead of real-time quantitative PCR, qPCR) on the low cost and ubiquity of the skills and equipment needed to deploy this technique (Xia et al 2018). The trade-off with this decision is that cPCR is nonquantitative and less sensitive than qPCR (Xia et al 2018).…”

“…We based our choice of conventional PCR (instead of real-time quantitative PCR, qPCR) on the low cost and ubiquity of the skills and equipment needed to deploy this technique (Xia et al 2018). The trade-off with this decision is that cPCR is nonquantitative and less sensitive than qPCR (Xia et al 2018). In addition, at the time of our analysis, no qPCR assays for dreissenids had been developed.…”

“…Based on labor, time, and consumables, we calculated the cost needed to process each sample and the labor required (Table 1). We have also included the cost of using qPCR in our results, as it has gained popularity in eDNA analyses (Xia et al 2018).…”

Identification of suitable reference genes for qPCR expression analysis on the gonads of the invasive mussel Limnoperna fortunei

“…To our knowledge, this is the first work that sought to validate RGs for the golden mussel in an effort to implement robust qPCR assays for gene expression analysis in this non-model organism. The application of qPCR to this species has been limited to the detection of larvae in the environment by absolute quantification, for which RGs are not required [38][39][40] and to one research article where qPCR was applied to evaluate gene expression in this mussel foot, but using a single non-validated RG [11]. We hope that the data presented here may help to change this picture, enabling the execution of reliable gene expression studies in the golden mussel gonads and contributing to advance the knowledge of the molecular mechanisms of key aspects of its extremely efficient reproduction, one of the distinguishing features of this aggressive invader.…”