Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

摘要

Quantitative real time PCR is a standard method for identification of species. One of the limitations of the qPCR is that there would be false-positive results due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (T_m) shifts. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by some factors. However, they are prevalently counterfeited or distributed without verification for getting higher prices. As it is almost impossible to find out visually the faked ones'31, it is becoming a social issue. Therefore, it is necessary to identify species at molecular level. In this research four species of squids, which have 1-2 base pair differences each, are selected as samples as they are affected by this issue. We designed two probes which are labeled with fluorescent dye and quencher at both ends of each probe. Two probes perfectly match with one species and have 1 or 2 base pair mismatch with other different two species respectively. In the experiment, we successfully distinguished three species with a single probe and four species with two probes by T_m shift depending on the difference of the base pairs. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, and furthermore, enabling analyses with smart phone would realize point-of-care testing (POCT).