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

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.

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

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

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