| Summary: | 碩士 === 輔英科技大學 === 醫事技術系碩士班 === 96 === Salmonellae are gram-negative bacilli belonging to the Enterobacteriaceae family. The clinical syndromes caused by the infection of Salmonella spp. vary to a large degree and may include gastroenteritis, bacteremia, and septicemia. Clinical diagnosis of Salmonella infection is on the basis of recovery and identification of bacteria from the specimens. Several kinds of enrichment broth have been applied to increase the recovery of Salmonella. In particular, SBG enrichment broth was demonstrated to increase the recovery in our previous study. The aim of this study is to investigate the method for molecular detection and identification of Salmonella spp.. To increase the recovery of Salmonella, SBG enrichment broth was included in this study. To detect Salmonella spp. in clinical samples with real-time PCR, the invA gene was chosen as the target gene. To avoid false-negative in entire process including DNA extraction and amplification steps, the M13 phage DNA was applied as an internal control. To further identify the serogroups of Salmonella spp. with molecular basis of O antigen, the specific primers of wzx genes for serogroup B and serogroup D were applied in the molecular identification with real-time PCR. The results demonstrated the performance of molecular detection of Salmonella with invA as a target gene and molecular serotyping of Salmonella with wzx gene in real-time PCR. First by, the M13 phage DNA was successfully applied as an internal control in the molecular detection of Salmonella with invA gene. Second by, following SBG enrichment the recovery of Salmonella from 241 clinical samples in by real-time PCR amplification of invA gene (46 positives) were 657% and 135% of those by direct-plating XLD (7 positives) and SBG-XLD (34 positives), respectively. Third by, the specificity of molecular detection of invA gene was demonstrated to be 100% with common pathogens and normal flora in enterogastric specimens. Fourth by, the limit of detection of invA gene was demonstrated to be 1 CFU (100%, 10/10). Fifth by, the molecular serotyping of 63 Salmonella strains based on wzx genes was demonstrated to be serogroup B (44/44), serogroup D (10/10), and non-B-non-D serogroup (9/9), respectively. The results were consistent with that obtained by serological method. Sixth by, the specificity of molecular serotyping of serogroup B and serogroup D were found to be 100%, by using with 2 species of common pathogens and 8 species of normal enteric flora found in enterogastric specimens. Seventh by, the limit of detection and of the wzx genes were 60% (6/10) at a concentration of 1 CFU and 100% (10/10) at a concentration of 10 CFU for both serogroup B and D. In conclusion, the study provides a useful method for detection and identification of Salmonella in clinical samples in terms of performance, sensitivity, and specificity.
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