Abstract
Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. Besides, the potential of qPCR to quantify DNA in samples makes it an invaluable molecular tool to infer the predation levels on specific prey by analysing predators’ stools. Nevertheless, studies on the diet of bats failed to find any empirical relationship, and it remains to be evaluated. Thus, we developed and evaluated two species-specific PCR assays to detect and quantify DNA of a major forest pest, the pine processionary, Thaumetopoea pityocampa, in bats’ faeces. Further, we empirically compared a range of different known DNA concentrations (input) of the target species mixed with mocks and bat faecal samples against DNA abundances yielded by qPCR (output) for a quantitative assessment. Overall, cPCR showed a lower detection rate than qPCR, but augmenting the replicate effort from one to three replicates led to a greater increase in the detection rate of the cPCR (from 57 to 80%) than the qPCR (from 90 to 99%). The quantitative experiment results showed a highly significant correlation between the input and output DNA concentrations (t = 10.84, p < 0.001) with a mean slope value of 1.05, indicating the accuracy of our qPCR assay to estimate DNA abundance of T. pityocampa in bat faeces. The framework of this study can be taken as a model to design similar assays applicable to other species of interest, such as agricultural pests or insects of public health concern.
Highlights
Targeted molecular methods such as conventional PCR and quantitative PCR, combined with species-specific primers and probes, are widely applied for pest species detection
Conventional PCR and real-time or quantitative PCR methods allow the amplification of minute amounts of template DNA even when the target is mixed with large amounts of non-target DNA, such as in animal scat[26]
Sequence analysis of the quantitative PCR (qPCR) products revealed that all the T. pityocampa amplicons were 100% identical to the expected 89 bp fragment (Fig. S2). conventional PCR (cPCR) products exclusively matched to T. pityocampa, but they did not show the same consistency as qPCR
Summary
Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. We developed and evaluated two species-specific PCR assays to detect and quantify DNA of a major forest pest, the pine processionary, Thaumetopoea pityocampa, in bats’ faeces. QPCR monitors the amplification process in real-time, and it provides a qualitative and quantitative assessment of the aimed sequence, but at a much higher cost Another determining factor is the number of replicates used. Increasing PCR replicates significantly improves the detection probability by reducing the risk of false negatives and yielding more reliable results[35] Making all these decisions (choosing PCR strategy or the number of replicates included) will involve a trade-off between the financial costs, logistical feasibility, and the risk of inaccurate results
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