Studies on quantitative gene probe development for pufferfish and genotoxicity of TTX

ASIAIR > College of Medical and Health Science > Department of Food Nutrition and Healthy Biotechnology > Theses & dissertations > Item 310904400/11581

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Title:	Studies on quantitative gene probe development for pufferfish and genotoxicity of TTX
Authors:	Chang,Hsiang-Yu
Contributors:	Department of Health and Nutrition Biotechnology
Keywords:	Puffer;Real-time quantitative PCR;TaqMan Probe
Date:	2011
Issue Date:	2011-09-30 01:12:25 (UTC+0)
Publisher:	Asia University
Abstract:	The majority 30 species of Taiwan pufferfish species are born with tetrodotoxin (TTX) and non-economic value due to small quantity of production. Due to fraud or incorrect manufacturing processes, different proportions of unexpected or pufferfish muscle may be incorporated. The volatility of these factors may lead to counterfeit and concerned issue for public food safety. To solve these problems, a rapid qualitative and quantitative method which can accurately and quantitatively authenticate the source fish species under different of production processing should be established as soon as possible. The first study was focused on the gene map analysis of cytochrome b (cyt b) and cytochrome c oxidase I (COI) for Lagocephalus genus and Takifugu genus. The results show the strong genus-specificity and sensitivity on cyt b gene of L. genus and COI gene of T. genus. The further designed genus specific primers and genus Taqman probes are LF-cytb74163 / LR-cytb86890 and T. genus: TF-COI31332 / TR-COI41233, L- probe cytb and T- probe COI of L. genus and T. genus respectively. The amplified length of fragments was 150 base pairs for L. genus and 121 base pairs for T. genus. Specificity and sensitivity of specific primers and genus Taqman probes were also tested. These results demonstrated the primers and Taqman probes were able to discriminate efficiently of L. and T. genus individually and did not have reaction with other species. The detection limit of DNA concentration is 10-2 ng/μL of L. genus and T. genus. Furthermore, apply the above method to identify the commercially products is successfully identify the dry-dressed fish fillets collected before which were all made from pufferfish. The recent collection of these two years from Taiwan’s northern, central, southern and eastern the samples were all not made from pufferfish. It was shown the primers and Taqman probes were able to identificate the processed products and coupled with Real-time PCR is an accurate qualitative analysis technique. Once the TTX was ingested into the body, the toxicity mechanism is blocking the sodium channels entry of sodium ion on neuronal membranes. Suppose low dose-TTX has been ingested that forms a number of different mononucleotide and dinucleotide adducts in DNA, TTX-derived DNA adducts may cause liver cell toxicity. This study actually provided a very important reference on TTX toxicogenomics analysis. In our study, the in vitro test of TTX interacted with a single nucleotide dGMP via the HPLC analysis was done. It was shown that TTX did not have reaction with dGMP. Our data can be used as the basic information of TTX toxicogenomics. To summarize our study actually provided a very important reference on quantitative analysis of sensitive and reliable analyzing method for food safety and especially for adulterated pufferfish products. In addition, the TTX of genotoxicity also provide the basis toxicogenomics information for future related research purposes, and this is also the first study. Our study actually proved groundbreaking and practicability on science research.
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