Salmonella serovar identification using PCR-based detection of gene presence and absence

Abstract

There are more than 2,500 known Salmonella serovars, and some of these can be further subclassified into groups of strains that differ profoundly in their gene content. We refer to these groups of strains as "genovars." A compilation of comparative genomic hybridization data on 291 Salmonella isolates, including 250 S. enterica subspecies I strains from 32 serovars (52 genovars), was used to select a panel of 384 genes whose presence and absence among serovars and genovars was of potential taxonomic value. A subset of 146 genes was used for real-time PCR to successfully identify 12 serovars (16 genovars) in 24 S. enterica strains. A further subset of 64 genes was used to identify 8 serovars (9 genovars) in 12 multiplex PCR mixes on 11 S. enterica strains. These gene panels distinguish all tested S. enterica subspecies I serovars and their known genovars, almost all by two or more informative markers. Thus, a typing methodology based on these predictive genes would generally alert users if there is an error, an unexpected polymorphism, or a potential new genovar.

FIG. 1.

Analysis of genetic differences (whole ORFs) between 32 Salmonella serovars (52 genovars). Black, no difference; gray, 1 difference; white, ≥2 differences. (A) Three hundred eight-four genes selected initially for real-time PCR. (B) One hundred forty-six genes reporting consistently in real-time PCR assays. (C) Sixty-four multiplexed genes scored on agarose gels. (D) Thirteen S. enterica serovar Typhimurium and serovar Typhi genes used in reference . Genovar numbers are shown on the top and the left side of each panel and correspond to column 1 of Table 1. Absence/presence data were obtained from microarray-based CGH analyses, as described in Materials and Methods. The number of differences in gene predictions (absence versus presence) was calculated in pairwise comparisons between model strains for each genovar. These model strains were those isolates with the best quality score of the hybridization within each genovar. The raw numbers of differences are shown in Fig. S1 in the supplemental material. Note: reference used an additional region specific to S. enterica serovar Enteritidis that was not sampled in our CGH experiments and therefore could not be included in this analysis.

FIG. 2.

Multiplex PCRs with 11 Salmonella enterica strains. Strain designations are shown in Table 3. Primer mix 10 is characterized in Table S1 in the supplemental material. L, DNA size standard.