2 June 2017 Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe
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Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result 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 (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).
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Eunjung Koh, Eunjung Koh, Ha Jeong Song, Ha Jeong Song, Na Young Kwon, Na Young Kwon, Gi Won Kim, Gi Won Kim, Kwang Ho Lee, Kwang Ho Lee, Soyeon Jo, Soyeon Jo, Sujin Park, Sujin Park, Jihyun Park, Jihyun Park, Eun Kyeong Park, Eun Kyeong Park, Seung Yong Hwang, Seung Yong Hwang, } "Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe", Proc. SPIE 10246, Smart Sensors, Actuators, and MEMS VIII, 102461L (2 June 2017); doi: 10.1117/12.2279779; https://doi.org/10.1117/12.2279779

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