Evaluation of gene markers invA, phsB, and tviA for detection of Salmonella Typhi by SYBR Green TM real-time PCR assay in clinical samples.

BACKGROUND: Diseases caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi remain a major public health concern. Due to overlapping clinical symptoms and the limitations of diagnostic methods, distinguishing S. Typhi from S. Paratyphi (A, B, and C) is challenging. Delayed and inaccurate diagnoses increase the risk of complications and transmission, highlighting the need for rapid, reliable molecular markers. AIM OF STUDY: This study aimed to develop and validate gene-based molecular markers, using optimized PCR conditions, for the accurate identification of S. Typhi in clinical samples. METHODS AND RESULTS: Primers targeting the invA, phsB, and tviA genes were designed and systematically assessed using conventional PCR to optimize thermal profiles and reaction conditions, followed by SYBR Green real-time PCR for rapid detection. Amplicon specificity was confirmed through gel electrophoresis, and invA amplicons were further validated by Sanger sequencing to confirm sequence conservation. The invA gene showed 90% amplification across all Salmonella isolates, confirming its reliability as a genus-level marker. Sanger sequencing verified the high conservation of the invA region. The phsB gene was amplified in 86.67% of S. Typhi isolates and 80% of S. Paratyphi isolates, indicating its potential as a phenotypic marker for H₂S production, a hallmark biochemical trait used to identify Salmonella. The tviA gene was detected in 86.67% of S. Typhi isolates and was absent in S. Paratyphi, validating its specificity for S. Typhi identification. Sequence alignment confirmed that the invA gene, encoding the InvA protein, is functionally conserved. The invA gene sequences of S. Typhi and S. Paratyphi are available at NCBI under the accession no PV704246.1 and PV704247.1, respectively. CONCLUSIONS: This study demonstrates that the invA, phsB, and tviA genes serve as effective molecular markers for the rapid and accurate diagnosis of S. Typhi.