Novel PCR assay for identification of Salmonella enterica serovar Infantis

Abstract

Aims: We developed, optimized and tested two novel PCR assays specific for Salmonella enterica subspecies enterica serovar Infantis. Methods and Results: The fljB gene was chosen as the target sequence. Primers were designed on a consensus sequence built by sequencing the fljB gene of five genetically unrelated Hungarian S. Infantis strains and using sequence data from the GenBank (http://www.ncbi.nih.gov). Two alternative assays were designed, which share the reverse primer. Both proved to be highly specific to S. Infantis, neither reacted with 42 other nontyphoidal serovariants tested. The detection limit of the assays was determined to be 105 CFU ml−1 from pure culture, and 106 CFU g−1 from artificially spiked chicken faeces samples. Conclusions: Although the detection limit is rather high to allow for using them for direct detection, the assays may be useful in identification of S. Infantis both for diagnostic and for research purposes. Significance and Impact of the Study: The described PCR assays allow for the correct identification of S. Infantis even when traditional serotyping methods fail because lack of expression of flagellar antigens.

Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis

“…Polymerase chain reaction (PCR) methods have shown promise in microbial diagnostics over the past decades because of their rapidity and higher accuracy compared to traditional antiserabased serotyping (Kardos, Farkas, Antal, Nogrady, & Kiss, 2007;Kim et al, 2009;Octavia & Lan, 2010;Perera & Murray, 2008;Trafny, Kozlowska, & Szpakowska, 2006). The detection specificity of PCR methods depends on the specific binding of the primer set to the target sequences in the organisms.…”

Development of PCR-based identification of Salmonella enterica serovars

“…Out of 64 isolates that were identifi ed as S. Infantis by conventional serotyping the same result was obtained for only 39 isolates (61 %) in simplex PCR. This was unexpected since selected primers were reported to be successfully used in S. Infantis identifi cation [18,[24][25][26][27]. The pair of primers used for identifi cation of S. Infantis serovar were designed to amplify practically the entire variable region of the fl agellar antigen fl jB gene.…”

“…All pairwise alignments were negative except for bcfC gene indicating that S. Infantis serovar did not have unique PCR profi le, but belonged to Group 2-type (Table 2). Therefore, to identify S. Infantis we have included a pair of primers specifi c for S. Infantis, developed by Kardos et al [18]. Similarly, other serovars whose genomes were not included in primers' design by Zhu et al [17], but identifi ed in our study by conventional serotyping (S. Derby, S. Havana, S. Livingstone, S. Mbandaka, and S. Senftenberg) were also in silico analyzed.…”

“…Oligonucleotides for the identifi cation of Salmonella enterica serovars used in this study were developed by Zhu et al [17] and Kardos et al [18]. We have tested a different number of pairs of primers per a PCR reaction (2 to 5 pairs) in various combinations, varied reaction compositions (different concentrations of dNTPs, enzyme, and primers), as well as PCR cycling protocols (ranges of annealing temperatures) using KAPA Taq PCR kit (KAPA Biosystems, USA).…”

“…To date the data on epizootic prevalence of different Salmonella serotypes in poultry on the territory of Serbia determined by molecular methods are scarce. Molecular serotyping scheme modifi ed to fi t our needs combined the work based on whole-genome sequencing and comparative genome analysis performed by Zhu et al [17] and study of Kardos et al [18] in order to identify S. Enteritidis and S. Infantis serotypes, and to cluster other Salmonella serovars in a way that enables their targeted identifi cation either by molecular or conventional identifi cation if necessary. To the best of our knowledge this study is the fi rst attempt to adapt a PCR protocol in order to identify Salmonella serovars in Serbia using molecular approach.…”