Qualitative and quantitative detection of Oreochromis niloticus-derived component 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 tilapia-derived components in fish meal, this study, we established a real-time fluorescence PCR method (RT-PCR) on detecting tilapia-derived components in fish meal by designing the tilapia-specific primers. The specificity of the primers was verified based on the DNA amplication of various fish, shrimps and crabs. Sensitivity of the method was detected basing on the examination of tilapia DNA gradient dilution template and different contents of tilapia component mixed with pure fish meal. Finally, the method was used to detected 8 different fish meal samples to evaluate whether there were tilapia-derived components in the samples. The results showed that the method had high specificity in identifying tilapia derived components. There was significant amplification signal only found in tilapia and no signal detected in other 50 common fish, shrimp and crab samples. In the sensitive experiment, when the diluted concentration of tilapia DNA was 0.1 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. In the experiment on detecting different content of tilapia component mixed with pure fish meal, when the mixing content of tilapia component was 0.1%, the Ct value was 33.87±0.49, when the content was 0.01%, the Ct value was 37.16±/A (>35), indicating that the method could detect the minimum concentration of tilapia DNA was 0.1 ng/μL and the minimum content of tilapia component was 0.1%. 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, revealing that certain tilapia 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 and they were judged as no tilapia components were detected. The result suggested that the method developed in this study can be used for rapid and effective detection of tilapia derived components in fish meal.