| Congresso Brasileiro de Microbiologia 2023 | Resumo: 521-2 | ||||
Resumo:Salmonella poses a significant challenge in poultry farming, making accurate diagnosis of this microorganism essential for ensuring animal health and product quality. While traditional microbiological diagnosis of Salmonella has been widely used and proven to be safe and effective, it can be time-consuming and labor-intensive. Molecular biology-based methods offer a viable and rapid alternative for detecting this pathogen. Isothermal amplification techniques, such as Loop-mediated isothermal amplification (LAMP), and variations of PCR, are routinely employed in laboratories for diagnosing salmonellosis due to their high sensitivity, specificity, and reduced analysis time. The objective of this study was to evaluate the application of LAMP and qPCR techniques in detecting Salmonella in chicken carcass samples. Genetic material extraction was conducted using magnetic beads, and primers based on the invA gene region were used for both techniques. For the LAMP reaction, 12.5 μL of WarmStart® LAMP 2X Master Mix (DNA & RNA), 1.6 μM of FIP and BIP primers, 0.2 µM of F3 and B3 primers, and 0.4 μM of Loop-F and Loop-B primers were mixed with 2 μL of the extracted sample and nuclease-free water to reach a total volume of 25 μL per reaction. The thermal incubation was carried out at 65ºC for 30 minutes, followed by 85ºC for 5 minutes. Samples that exhibited green fluorescence under UV light were identified as positive. In the qPCR reaction, 10 µL of GoTaq® qPCR Master Mix (Promega, Madison, USA), 0.6 µL of each primer (10 µM), 5 µL of extracted DNA, and nuclease-free water were mixed to obtain a total volume of 20 µL. Thermocycling included an initial step at 95ºC for 5 minutes, followed by 40 cycles of 95ºC for 15 seconds and 60ºC for 1 minute, concluding with a melting curve analysis. The study analyzed 33 chicken carcass samples, which were also subjected to conventional microbiological diagnosis at the Laboratório de Inspeção Sanitária de Alimentos do SOAP/FMVZ/UNESP/Botucatu. Among the samples, 7 out of 33 (21%) exhibited amplification in qPCR, and 4 of these were also detectable in LAMP and conventional microbiology. Two samples were detectable only in qPCR and did not have their presence confirmed by conventional microbiology, while 1 sample was detectable in both LAMP and qPCR, but its presence was not confirmed by conventional microbiology. The results obtained from the study using LAMP and qPCR techniques demonstrated their effectiveness in specifically detecting Salmonella. The invA gene was chosen as the target for molecular detection due to its broad specificity for various Salmonella serovars and its ability to distinguish non-Salmonella strains. The evaluation of these molecular biology techniques alongside conventional microbiology revealed agreement in the results. Only 1 sample showed discordance between the microbiological and molecular results (LAMP and qPCR), and 2 samples were detectable only in qPCR. These findings may be attributed to the limitations of the qPCR technique, as it amplifies genetic material without the ability to differentiate between viable and non-viable cells. In conclusion, the detection and continuous monitoring of Salmonella are crucial for preserving animal health, safeguarding public health, and ensuring poultry production. Palavras-chave: Salmonella, Isothermal amplification, Rapid detection, Chicken carcasses | |||||