Molecular based method for the detection and quantification of larvae of the golden mussel Limnoperna fortunei using real-time PCR

Abstract

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.