Abstract
Larimichthys polyactis (redlip yellow croaker) and Larimichthys crocea (large yellow croaker) are commercially important fish species in East Asia with high differences in their market values. In Korea, consumers prefer L. polyactis to L. crocea, although it is difficult to distinguish them based on their morphological traits. The objective of this study was to develop an assay for differentiating L. polyactis and L. crocea using fluorescence melting curve analysis (FMCA) with a single locked nucleic acid (LNA) probe. Species-specific regions of the mitochondrial 16S rDNA were selected as LNA probes. The target sequences of L. polyactis and L. crocea had a 2-bp difference, and a single LNA probe was identified using melting temperature (Tm) shift. LNA probe was 100 % complementary to the target sequence of ten L. polyactis samples, giving a significantly higher Tm value (66 °C) than that of five L. crocea samples (42 °C). Overall, the developed LNA-based FMCA system had high efficiency, multiplexity, and simplicity, and could be effectively used for differentiating L. polyactis and L. crocea, and as a food analyzing method based on DNA sequence.
References
Ahn JJ, Kim J-H, Kim Y, Hong JY, Kim GW, Hwang SY (2015) Multiplex genoty** based on the melting point of a single locked nucleic acid probe Analytical biochemistry
Aranishi F (2005) Rapid PCR-RFLP method for discrimination of imported and domestic mackerel. Mar Biotechnol 7:571–575
Bernard PS, Pritham GH, Wittwer CT (1999) Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genoty**. Anal Biochem 273:221–228
Braasch DA, Corey DR (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 8:1–7
Cho H, Kim J-H, Lee WS, Jeong W, Moon H-B, Hwang SY (2014) Development of molecular detection kit for Larimichthys crocea and Larimichthys polyactis. BioChip J 8:148–153
Elenitoba-Johnson KS, Bohling SD, Wittwer CT, King TC (2001) Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis. Nature Med 7:249–253
Floren C, Wiedemann I, Brenig B, Schütz E, Beck J (2015) Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR). Food Chem 173:1054–1058
GrÜnweiler A, Hartmann RK (2007) Locked nucleic acid oligonucleotides. BioDrugs 21:235–243
Gunson R, Bennett S, Maclean A, Carman W (2008) Using multiplex real time PCR in order to streamline a routine diagnostic service. J Clin Virol 43:372–375
Huang Q, Liu Z, Liao Y, Chen X, Zhang Y, Li Q (2011) Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes. PLoS One 6:e19206
Jacobsen N, Bentzen J, Meldgaard M, Jakobsen MH, Fenger M, Kauppinen S, Skouv J (2002) LNA‐enhanced detection of single nucleotide polymorphisms in the apolipoprotein E. Nucleic Acids Res 30:e100
Johnson MP, Haupt LM, Griffiths LR (2004) Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real‐time PCR. Nucleic Acids Res 32:e55
Kesmen Z, Gulluce A, Sahin F, Yetim H (2009) Identification of meat species by TaqMan-based real-time PCR assay. Meat Sci 82:444–449
Latorra D, Campbell K, Wolter A, Hurley JM (2003) Enhanced allele-specific PCR discrimination in SNP genoty** using 3′locked nucleic acid (LNA) primers. Hum Mutat 22:79
Martinez I, Jakobsen Friis T (2004) Application of proteome analysis to seafood authentication. Proteomics 4:347–354
Ørum H, Jakobsen MH, Koch T, Vuust J, Borre MB (1999) Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids. Clin Chem 45:1898–1905
Rasmussen RS, Morrissey MT (2008) DNA‐based methods for the identification of commercial fish and seafood species. Compr Rev Food Sci Food Saf 7:280–295
Teletchea F (2009) Molecular identification methods of fish species: reassessment and possible applications. Rev Fish Biol Fish 19:265–293
Ugozzoli LA, Latorra D, Pucket R, Arar K, Hamby K (2004) Real-time genoty** with oligonucleotide probes containing locked nucleic acids. Anal Biochem 324:143–152
Watanabe S, Minegishi Y, Yoshinaga T, Aoyama J, Tsukamoto K (2004) A quick method for species identification of Japanese eel (Anguilla japonica) using real-time PCR: An onboard application for use during sampling surveys. Mar Biotechnol 6:566–574
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This study was supported by the National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology, Republic of Korea (No. 2012R1A2A2A03045008)
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Ahn, J.J., Kim, Y., Hong, J.Y. et al. Probe-Based Fluorescence Melting Curve Analysis for Differentiating Larimichthys polyactis and Larimichthys crocea . Food Anal. Methods 9, 2036–2041 (2016). https://doi.org/10.1007/s12161-015-0381-6
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DOI: https://doi.org/10.1007/s12161-015-0381-6