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KMID : 0613820170270111331
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Journal of Life Science
2017 Volume.27 No. 11 p.1331 ~ p.1339
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Development and Validation of Real-time PCR to Determine Branchiostegus japonicus and B. albus Species Based on Mitochondrial DNA
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Chung In-Young
Seo Yong-Bae
Yang Ji-Young
Kim Kun-Do
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Abstract
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DNA barcoding is the identification of a species based on the DNA sequence of a fragment of the cytochrome C oxidase subunit I (COI) gene in the mitochondrial genome. It is widely applied to assist with the sustainable development of fishery-product resources and the protection of fish biodiversity. This study attempted to verify horse-head fish (Branchiostegus japonicus) and fake horse-head fish (Branchiostegus albus) species, which are commonly consumed in Korea. For the validation of the two species, a real-time PCR method was developed based on the species' mitochondrial DNA genome. Inter-species variations in mitochondrial DNA were observed in a bioinformatics analysis of the mitochondrial genomic DNA sequences of the two species. Some highly conserved regions and a few other regions were identified in the mitochondrial COI of the species. In order to test whether variations in the sequences were definitive, primers that targeted the varied regions of COI were designed and applied to amplify the DNA using the real-time PCR system. Threshold-cycle (Ct) range results confirmed that the Ct ranges of the real-time PCR were identical to the expected species of origin. Efficiency, specificity and cross-reactivity assays showed statistically significant differences between the average Ct of B. japonicus DNA (21.85±3.599) and the average Ct of B. albus DNA (33.49±1.183) for confirming B. japonicus. The assays also showed statistically significant differences between the average Ct of B. albus DNA (22.49±0.908) and the average Ct of B. japonicus DNA (33.93±0.479) for confirming B. albus. The methodology was validated by using ten commercial samples. The genomic DNA-based molecular technique that used the real-time PCR was a reliable method for the taxonomic classification of animal tissues.
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KEYWORD
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Branchiostegus albus, Branchiostegus japonicas, mitochondrial DNA, real-time quantitative PCR
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