| Abstract: | The five billfishes are the important economical fish in Taiwan. They are used in many kinds of products, including slices of meat (sashimi), fish minced products and dried fish floss. The meats and products have a relatively high market price in Taiwan. Therefore, some retail dealers misuse the other low-price fish meats instead of billfish meats in order to make a profit.
In this study we used of a specific Real-time PCR amplification of a repetitive DNA element, for the quantification by billfishes, because of its simplicity, specificity, and sensitivity.
In the beginning we amplified of cycochrome c oxidase I and 16S rRNA finished the complete of billfishes sequencing, and designed suitable primers of DNA quantitative method of Real-time PCR.
In this study, we used five billfishes assays were developed around small (amplicons 150 base pairs) region of the mitochondrial cytochrome c oxidase I (COI) gene. The assay first was tested on specificity and sensitive of primers. We defined that the primers were applicable to the five billfish, so second we used the primers detect the limit of billfish concentration, the sensitivity was as following: Makaira nigricans: 10-5 ng, Kajikia audaxt: 10-5 ng, Istiophorus platypterus: 10-4 ng, Xiphias gladius: 10-3 ng and Istiompax indica: 10-5 ng.
Third was test on different time of heated and sterilized ( 100℃ heating from 0 – 90 min, 121℃ heating from 0 – 90 min) to define the limit of five billfish. The results showed the heted of billfish meats limit detected time was > 60 min, and the sterilized detected limit time was >30 min. Forth the assay was tested on DNA extracted from raw and heat-treated mixtures of billfishes tissues in a matrix, and on DNA extracted from reference stuff samples.The mixture of billfish meats limit detected was <0.5% of billfish’s DNA concentration, and the related products of billfish limit detected was about <1%. Replicate standard curves of the threshold cycle (Ct) value (Y) vs Different percentages of billfish DNA (X) were analysed using sigama plot 10.0 software and a linear regression equation of the Ct value plotted against Different percentages of billfish’s DNA was calculated.
Furthermore, applying above the method to quantitative of billfish, we collected 44 commerical samples of billfish related products from northern region, centeral region, southern region and eastern region. It was found that qutitative of billfish in dressed fish fried marlin product were 30.47% - 100%, fish ball were 14.52% - 93.45%, surimi made by marlin was 87.21%, tempura made by marlin was 74.94%, and marlin egg roll was 71.03%.
Base on the results we concluded that Real-time PCR condition developed in our research was applicable to the quantitative analysis of detecting the marlin meat products. |
