Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun;103(12):4987-4996.
doi: 10.1007/s00253-019-09854-4. Epub 2019 May 6.

Development of a real-time PCR method for the genoserotyping of Salmonella Paratyphi B variant Java

Mathieu Gand  1   2 , Wesley Mattheus  3 , Assia Saltykova  2   4 , Nancy Roosens  4 , Katelijne Dierick  5 , Kathleen Marchal  2   6 , Sigrid C J De Keersmaecker  4 , Sophie Bertrand  7
Affiliations

Development of a real-time PCR method for the genoserotyping of Salmonella Paratyphi B variant Java

Mathieu Gand et al. Appl Microbiol Biotechnol. 2019 Jun.

Abstract

Discriminating between D-tartrate fermenting and non-fermenting strains of Salmonella enterica subsp. enterica serotype Paratyphi B is of major importance as these two variants have different pathogenic profiles. While D-tartrate non-fermenting S. Paratyphi B isolates are the causative agent of typhoid-like fever, D-tartrate fermenting isolates (also called variant Java) of the same serotype trigger the less dangerous gastroenteritis. The determination of S. Paratyphi B variants requires a time-consuming process and complex biochemical tests. Therefore, a quadruplex real-time PCR method, based on the allelic discrimination of molecular markers selected from the scientific literature and from whole genome sequencing data produced in-house, was developed in this study, to be applied to Salmonella isolates. This method was validated with the analysis of 178 S. Paratyphi B (D-tartrate fermenting and non-fermenting) and other serotypes reaching an accuracy, compared with the classical methods, of 98% for serotyping by slide agglutination and 100% for replacement of the biochemical test. The developed real-time PCR permits to save time and to obtain an accurate identification of a S. Paratyphi B serotype and its D-tartrate fermenting profile, which is needed in routine laboratories for fast and efficient diagnostics.

Keywords: D-tartrate; Identification; Paratyphi B; Real-time PCR; Salmonella; WGS.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Presence of the marker SPAB_01124 in the S. Paratyphi B genomes. Alignment of the primer pPB23 (used in PCR Zhai and based on the marker SPAB_01124) against the multiple alignment of the de novo assembled in-house sequenced S. Paratyphi B genomes and the 16 publicly available S. Paratyphi B genomes using Bionumerics
Fig. 2
Fig. 2
Alignment of primers and probes designed for marker SPAB_04460 against sequences of the serotypes mentioned in Table 4. The designed primers (ParaB Fw and Rv) are amplifying a fragment of 79 bp. The probe ParaB contains in the middle of its sequence the SNP specific for S. Paratyphi B. The SNPs located in the annealing sites of some serotypes did not affect the efficiency of the qPCR assay, as they were not in the 3′ end of the primer nor in the middle of the TaqMan probes
Fig. 3
Fig. 3
Proposed analysis process for S. Paratyphi B dT−/dT+ identification in routine laboratories. In case of Other Salmonella, the full antigenice formula is determined by classical method. ae.g. S. Stanleyville. be.g. S. Berta, S. Meleagridis, and S. Singapore
See this image and copyright information in PMC

References

    1. Achtman M, Wain J, Weill FX, Nair S, Zhou Z, Sangal V, Krauland MG, Hale JL, Harbottle H, Uesbeck A, Dougan G, Harrison LH, Brisse S, S. enterica MLST Study Group Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog. 2012;8(6):e1002776. doi: 10.1371/journal.ppat.1002776. - DOI - PMC - PubMed
    1. Agren J, Sundstrom A, Hafstrom T, Segerman B. Gegenees: fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups. PLoS One. 2012;7:e39107. doi: 10.1371/journal.pone.0039107. - DOI - PMC - PubMed
    1. Alfredsson GA, Barker RM, Old DC, Duguid JP. Use of tartaric acid isomers and citric acid in the biotyping of Salmonella Typhimurium. J Hyg. 1972;70(4):651–666.1. doi: 10.1017/S0022172400022518. - DOI - PMC - PubMed
    1. Barbeau-Piednoir E, Botteldorn N, Yde M, Mahilon J, Roosens NH. Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes. Appl Microbiol Biotechnol. 2013;97(9):4021–4037. doi: 10.1007/s00253-012-4477-2. - DOI - PMC - PubMed
    1. Barbeau-Piednoir E, Bertrand S, Mahilon J, Roosens NH, Botteldorn N. SYBR® Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels. Appl Microbiol Biotechnol. 2013;97:9811–9824. doi: 10.1007/s00253-013-5234-x. - DOI - PMC - PubMed

LinkOut - more resources