A molecular based method for the quantitative detection of larvae of the golden mussel Limnoperna fortunei was developed. Partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene in several bivalve species including L. fortunei were analyzed. These nucleotide sequences and the CO1 gene for bivalve species found in habitats similar to L. fortunei (obtained from the EMBL/Genbank/DDBJ databases) were aligned. Based on these results, L. fortunei-specific primers were designed for amplification. DNA extracted from several bivalve species including L. fortunei were subjected to PCR using L. fortunei-specific primers, and amplification of the DNA fragment was confirmed only in PCR assays which utilized template DNA from L. fortunei. No DNA fragments were amplified during the PCR of freshwater planktonic sample lacking golden mussel larvae which were collected from Lake Ohshio (Gunma, Japan). Real-time PCR was performed using fluorescent dye (SYBR Green) detection. Threshold cycles correlated well with the template DNA and number of larvae equivalents, and the R-square values of the standard curves were 0.99 and 0.98, respectively. These results suggest that this real-time PCR-based method can be utilized not only for the identification of L. fortunei in a particular habitat, but also for the quantification of larvae of this species in natural environments.

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

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