Establishment of methods for the detection of Salmonella species by conventional and quantitative real-time PCR

The aim of this study was to establish a highly specific and sensitive method to detect Salmonella spp. based on conventional PCR and quantitative real-time PCR (qPCR) and to validate its application in clinical and animal samples. First, primers and probes specific to the STM1410 gene were designed and systematically optimized in terms of annealing temperature, primer concentration, and probe concentration. Then, the method was used to test clinical samples to evaluate its specificity, stability, and sensitivity. The results showed that both PCR and qPCR successfully detected the genomes of 15 different serotypes of Salmonella. In contrast, no detection was observed in the genomes of eight other common pathogens or in negative controls, confirming the high specificity of the methods. The minimum detection limit of conventional PCR was 5 × 10¹ copies/μL, and the minimum detection limit of qPCR was 5 × 10° copies/μL, both of which are highly sensitive. In the qPCR reproducibility test, the coefficients of variation (CVs) were <3% for both intra-group and inter-group analyses, demonstrating excellent stability. Finally, 60 clinical samples were tested using the method established in this study. The results showed that the positive rates were 29/60 (48.3%) for conventional PCR, 32/60 (53.3%) for TaqMan qPCR. The study developed conventional PCR and TaqMan qPCR methods targeting a Salmonella spp.-specific gene sequence, the STM1410 gene. These methods can be effectively used for clinical detection and food hygiene monitoring, thus holding great significance for promoting healthy farming practices and ensuring food safety.