Development and application of Real-Time PCR for the detection of important gastropod and Natica lineate (toxic species) in Taiwan

[[abstract]]Gastropod is one of the most common seafood resources in Taiwan. Most of the time, the gastropods shell were removed before processing. When the processed gastropod products finished, it is difficult to identify the species. Hence, as a result of losing distinguishable exterior characteristics, other identification approaches are needed for gastropod species forensics. Due to the severe processed procedures, protein is cleaved. Therefore, this study adopts genetic technologies for identification. This study is to perform gene map analysis of partial sequence cytochrome b (cyt b) for 8 gastropods and Natica lineate in Taiwan. The result shows that the species-specificity and sensitivity of the target DNA on cyt b Natica lineate species gastropod. The newly designed species-specific primers and TaqMan probe are Natica lineate F/ Natica lineate R and Natica lineate P of Cyt b in Natica lineate. The amplified length of fragment was 119 base pairs for Natica lineate (include Natica lineate F/ Natica lineate R primers). Specificity and sensitivity of specific primers and TaqMan probe were also tested. These results demonstrated that the primers and TaqMan probe were able to distinguish Natica lineate species from the other gastropod species. The detection limit of DNA concentration is 0.01 ng/μL of Natica lineate. Furthermore, the application of above method is to identify the commercially gastropod products. Fluorescent profiles of Real-time PCR products were amplified from Natica lineate muscles with specific probe, of which the muscles had been autoclaved at 121°C for 20 minutes to 100 minutes. The result shows that recent collection of market samples were without made by Natica lineate. It was shown that the primers and TaqMan probes were able to identify the processed products and were coupled with Real-time PCR is an accurate qualitative analysis technique