Evaluation of mdh, dld, tcfA, and folE gene markers for detection of enteric fever using real-time PCR

Enteric fever, a systemic infection caused by Salmonella enterica serovars Typhi and Paratyphi, continue to pose a substantial public health concern, with an estimated 20 million cases annually. It is transmitted via the consumption of contaminated food or water and is a substantial public health issue in low- and middle-income nations, resulting in extended fever, stomach pain, and severe sequelae if not addressed. The clinical diagnosis of enteric fever is complicated by nonspecific symptoms, the low sensitivity and specificity of blood cultures and serological methods, and the restricted application of qPCR in routine diagnostics, partly due to the insufficient testing or validation of numerous gene targets across various Salmonella strains. The study was conducted to evaluate mdh, dld, tcfA, and folE gene markers for detection of enteric fever causing typhoidal Salmonella species using real-time PCR. Primers specific to the mdh, dld, tcfA, and folE genes were developed and assessed utilizing Primer3Plus, an oligo calculator, and NCBI BLAST. Conventional PCR with optimized thermal profiles was employed for amplification, succeeded by SYBR Green-based real-time PCR and gel electrophoresis to verify the size and specificity of the amplicons. Sanger sequencing of specific folE amplicons confirmed their identity and revealed conserved sequences among S. Typhi and S. Paratyphi. The mdh gene demonstrated significant genus-level specificity, whereas dld and tcfA exhibited diagnostic potential for typhoidal strains. Initial in-silico predictions suggested that folE was specific to S. Typhi, but it was also experimentally identified in S. Paratyphi isolates. Sanger sequencing validated significant sequence similarity and a synonymous SNP, demonstrating functional conservation of folE, which encodes GTP cyclohydrolase I, among typhoidal Salmonella. The folE gene sequences for S. Typhi and S. Paratyphi have been submitted to GenBank with accession numbers PV700621 and PV700622. The assay demonstrated a strong correlation with biochemical findings; however, its restricted capacity to differentiate between typhoidal serovars underscores the necessity for more specific molecular markers to enhance diagnostic accuracy, inform targeted therapy, and facilitate comprehensive epidemiological surveillance.