Qualitative and quantitative detection of Oreochromis niloticus-derived components in fish meal based on fluorescent quantitative PCR

Fish meal is one of the important raw materials in aquatic feed. It has complete amino acid composition, high calcium, phosphorus and vitamin content, which is widely used in feed production. Recently, its large use and high price lead to the serious adulteration in fish meal market, tilapia (Oreochromis niloticus) is one of the common adulterated raw materials. In order to quickly and accurately detect O. niloticus-derived components in fish meal, this study, we established a real-time fluorescence PCR method (RT-PCR) on detecting O. niloticus-derived components in fish meal by designing the O. niloticus-specific primers. The specificity of the primers was verified based on the DNA amplification of various fish, shrimps and crabs. Sensitivity of the method was detected based on the examination of O. niloticus DNA gradient dilution template and different contents of O. niloticus component mixed with pure fish meal. Finally, the method was used to detect 8 different fish meal samples to evaluate whether there were O. niloticus-derived components in the samples. The results showed that the method had high specificity in identifying O. niloticus-derived components. There was significant amplification signal only found in O. niloticus and no signal detected in other 50 common fish, shrimp and crab samples. In the sensitive experiment, when the diluted concentration of O. niloticus DNA was 0.100 0 ng/μL, RT-PCR showed a typical specific amplification curve with a Ct value 32.60±0.36, when the diluted concentration was 0.01 ng/μL, the Ct value was 35.01 ± 0.26, which was larger than the Ct value of 35.00. In the experiment on detecting different content of O. niloticus component mixed with pure fish meal, when the mixing content of O. niloticus component was 0.1%, the Ct value was 33.87±0.49, when the content was 0.010 0%, the Ct value was 37.16±N/A (>35.00), indicating that the method could detect the minimum concentration of O. niloticus DNA was 0.100 0 ng/μL and the minimum content of O. niloticus component was 0.100 0%. In detecting 8 different fish meal samples from the market basing on this method, 4 fishmeal samples: Pakistan fishmeal, Myanmar fishmeal, Vietnamese fishmeal and domestic fishmeal 2 were detected to exist significant amplification signals and their Ct values were all lower than 35.00, revealing that certain O. niloticus components were mixed in those 4 samples. The Ct value of Peruvian fishmeal, Japanese fishmeal, domestic fishmeal 1 and imported super fishmeal were larger than 35.00 and they were judged as no O. niloticus components were detected. The result suggested that the method developed in this study can be used for rapid and effective detection of O. niloticus-derived components in fish meal.