Desenvolvimento de sistema de detecção molecular para Piracatinga Calophysus macropterus (Lichtenstein, 1819) e Dourada Brachyplatystoma rousseauxii (Castelnau,1855) e validação em filés de pescados da região amazônica

Abstract

Fish consumption has increased exponentially over the last few decades, boosting trade and making a profit for the fishing industry. In this way, fish is supplied to the consumer in various ways, whether fresh, in slices, canned, or filleted. However, despite their practicality and speed of preparation, fish sold in filleted formats no longer have enough morphological characteristics to recognize the species to which they belong, which can lead to food fraud through species substitution. Some studies have already used molecular biology tools to identify commercial fraud. The most common include generating nucleotide sequences obtained by extracting, amplifying, and sequencing DNA from fillets, but this technique proves to be costly as it requires more infrastructure to be carried out. It is, therefore, important to develop a method that can be effective and cost-effective for this application. This study aimed to generate two species-specific molecular detection systems (MDS) involving two Amazonian fish species, one of which is used for fraudulent purposes (Calophysus macropterus) and the other which is of great commercial importance (Brachyplatystoma rousseauxii) in the Northern Region. This method involves using a hydrolysis probe for real-time amplification, commonly referred to as qPCR (Quantitative PCR), and, due to the sensitivity of the method, it can detect minute amounts of DNA in a sample with high specificity. To this end, a molecular detection system was developed using the mitochondrial DNA control region for sea bream (Brachyplatystoma rousseauxii) and the cytochrome oxidase subunit I (COI) gene for piracatinga (Calophysus macropterus), both of which were validated in qPCR tests with a hydrolysis probe, both in silico and in vitro, and fish sold in fillet form. Initially, tests were carried out using the probe and DNA from the study's target species and congeneric species to verify the probe's performance in amplifying only the target species. In subsequent stages, the fillet packages to be tested were chosen at random, and those fillet packages whose labels had been previously confirmed by the DNA barcoding methodology were included in these tests. The results show that fraud was detected in packages labeled "Dourada", as well as the presence of Calophysus macropterus in these products. This tool has shown great efficiency in detecting the target species and has a relevant economic, medical, and conservationist bias, as its usefulness can help the fishing sector to market products involving these two species of fish with greater safety and quality.