This application note describes a rapid, robust and easy to use protocol for identification of sturgeon species based on a well established PCR-RFLP method. The improved method allows analysis from sample to result in one working day and yields good discrimination results.
This document details a method for the discrimination of sturgeon and related species using PCR-RFLP analysis on the Agilent 2100 Bioanalyzer system. The protocol was developed to address the high homology between sturgeon species and to facilitate the identification of species from processed food samples, such as caviar roe.
The study evaluates the use of the Agilent DNA Fish ID solution with modified enzymes, specifically replacing Dde I with Csp6 I, to generate distinguishable restriction fragment patterns. The text provides detailed materials and methods for DNA isolation from fish tissue and roe, PCR amplification of the Cytb target sequence, and subsequent restriction digestion. Results demonstrate that the modified protocol allows for the grouping of various sturgeon species into distinct clusters based on fragment sizes.
| Tissue sample size for DNA isolation | 50 mg to 100 mg of tissue or 30 to 40 mg roe |
|---|---|
| Proteinase K digestion temperature | 65 °C |
| Restriction digestion duration | 2 h |
| DNA elution temperature | 70 °C |
| Chip type for analysis | DNA 1000 chip |
The protocol is designed to identify sturgeon species from fish eggs in caviar shipments.
The protocol uses fifty mg to 100 mg of muscle tissue or fin clippings, or thirty to forty mg of roe.
Digestion is performed at 65 °C for forty minutes with vortexing three times during incubation.
Four independent digests are loaded in consecutive wells to allow analysis of three samples per chip.