Development and validation of a real-time PCR and amplicon sequencing method for discriminating <i>Salmonella enterica</i> serovar Typhi in environmental samples

ABSTRACT The figure displays a flowchart detailing the proposed process for identifying Salmonella enterica serovar Typhi in environmental samples: (1) sample collection; (2) sample processing; (3) real-time PCR; (4) amplicon sequencing and single-nucleotide polymorphism confirmation; and (5) identification of the pathogen. Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, is notoriously difficult to identify in environmental samples (e.g., water, wastewater, and sewage runoff), due to an overabundance of Salmonella spp. and inadequately selective culture methods. To bypass difficulties with isolate recovery, a two-step molecular method for directly identifying S. Typhi in environmental samples was developed, utilizing an initial real-time polymerase chain reaction (qPCR) screening for presumptive S. Typhi identification followed by sequencing of qPCR products for confirmation. The qPCR assay exhibited a limit of detection of 63.8 gene copies per reaction and a limit of quantification of 638 copies per reaction. When tested against 44 S. enterica serovars, amplification occurred in S. Typhi and 7 non-Typhi serovars (Bareilly, Irumu, Lomalinda, Manchester, Moero, Napoli, and Oslo), while 36 of 43 nontarget serovars did not amplify, corresponding to 83.7% specificity. Amplicons were Sanger sequenced, and Typhi was differentiated from nontyphoidal serovars by a single nucleotide substitution identified in silico. In seeded water concentrates, the assay detected as low as 5 × 102 CFU/mL, demonstrating utility in complex environmental matrices.