Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

Abstract

Aims: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. Methods and Results: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC‐d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which coamplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 104 CFU ml−1 (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre‐enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. Conclusions: The developed mPCR assay provides specific detection of S. Typhi. Significance and Impact of the Study: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.

Detection of hemolytic strains of Aeromonas hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products by multiplex PCR

“…There are several conventional as well as rapid systems including PCR that have been tried for the detection of seafood borne pathogens (Kumar et al 2006;Jeyasekaran et al 2011;Raj et al 2011). Although Aeromonas spp.…”

Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay

“…No false positives or false negatives were recorded. IAC was included as the integral component of the assay in order to make the mPCR assay acceptable to the present norms of diagnostic tool [16]. The IAC incorporated was pUC 18 plasmid DNA fl anked by aerA primers.…”

“…Numerous methods, both the conventional as well as rapid systems including PCRs have been tried for the detection of foodborne pathogens [13,16,18]. However, in the case of Aeromonas, the PCR has mostly been used for the characterization of isolates either for their virulence properties or for the phylogenetic position [11,23].…”

“…The concentration of IAC DNA was determined spectrophotometrically at 260 nm and was stored in DDW. The following equation was used to calculate the copy number of the PCR product concentration: weight of PCR fragment (in g μl -1 ) × (6.023 × 1023)/(660 g mol -1 × number of base pairs of PCR fragment) = the number of genomic copy per microlitre [16].…”

Perfil epidemiológico e caracterização molecular de Salmonella Typhi isoladas no Estado do Pará, Brasil

“…A demora no diagnóstico inviabiliza a conduta terapêutica desejada, constituindo fator agravante diante da severidade clínica que alguns casos podem assumir. Uma forma de contornar esse problema é desenvolver ensaios baseados na amplificação de DNA, os quais, futuramente, poderão ter utilização rotineira, beneficiando o usuário do sistema por encurtar o tempo para o diagnóstico e pela precisão e confiabilidade na identificação do agente, facilitando a atuação das autoridades de saúde nas ações de controle do agravo 12,13 .…”