Assessing the efficacy of a quantitative PCR screening procedure for detecting and quantifying the magnitude of invasive carp reproductive events

ABSTRACT Objective A quantitative PCR (qPCR) screening procedure previously showed potential for rapidly assessing the likelihood that ichthyoplankton samples contain bigheaded carp Hypophthalmichthys spp. eggs and larvae. We sought to expand the capabilities of this procedure, assess its sensitivity under realistic field conditions, investigate potential sources of error, and compare the costs of this method with those of traditional microscopy-based sample processing. Methods We refined the qPCR procedure by screening for two additional species of invasive carp, tested whether the quantity of organic debris within ichthyoplankton samples influenced the procedure’s accuracy, assessed whether qPCR results could predict the number of target eggs and larvae in a sample, and compared the costs of the qPCR method to those of conventional sample processing. Results Invasive carp DNA copy number was a significant predictor of the presence of invasive carp specimens and had a sigmoidal relationship with the numbers of invasive carp eggs and larvae within a sample. The number of Silver Carp H. molitrix and Grass Carp Ctenopharyngodon idella DNA copies in a sample was also positively related to the number of specimens of each of these species in the sample. A 20% probability threshold of inferring a positive detection based on DNA copy number provided an optimal balance between sensitivity and specificity. High quantities of organic debris within samples increased the probability of obtaining a false-positive result across a range of detection thresholds. Additionally, high quantities of organic matter were associated with overestimation of the number of specimens predicted to occur within samples. The qPCR procedure required less time but incurred a higher monetary cost per sample than manual processing unless a moderate number of specimens was sequenced following visual identification. Conclusions Application and further modification of this qPCR procedure may allow for early detection or more detailed study of spawning by any or all invasive carp species or other pelagic broadcast spawners.