Molecular determination of H antigens of Salmonella by use of a microsphere-based liquid array

Abstract

Serotyping of Salmonella has been an invaluable subtyping method for epidemiologic studies for more than 70 years. The technical difficulties of serotyping, primarily in antiserum production and quality control, can be overcome with modern molecular methods. We developed a DNA-based assay targeting the genes encoding the flagellar antigens (fliC and fljB) of the Kauffmann-White serotyping scheme. Fifteen H antigens (H:a, -b, -c, -d, -d/j, -e,h, -i, -k, -r, -y, -z, -z(10), -z(29), -z(35), and -z(6)), 5 complex major antigens (H:G, -EN, -Z4, -1, and -L) and 16 complex secondary antigens (H:2, -5, -6, -7, -f, -m/g,m, -m/m,t, -p, -s, -t/m,t, -v, -x, -z(15), -z(24), -z(28), and -z(51)) were targeted in the assay. DNA probes targeting these antigens were designed and evaluated on 500 isolates tested in parallel with traditional serotyping methods. The assay correctly identified 461 (92.2%) isolates based on the 36 antigens detected in the assay. Among the isolates considered correctly identified, 47 (9.4%) were partially serotyped because probes corresponding to some antigens in the strains were not in the assay, and 13 (2.6%) were monophasic or nonmotile strains that possessed flagellar antigen genes that were not expressed but were detected in the assay. The 39 (7.8%) strains that were not correctly identified possessed an antigen that should have been detected by the assay but was not. Apparent false-negative results may be attributed to allelic divergence. The molecular assay provided results that paralleled traditional methods with a much greater throughput, while maintaining the integrity of the Kauffmann-White serotyping scheme, thus providing backwards-compatible epidemiologic data. This assay should greatly enhance the ability of clinical and public health laboratories to serotype Salmonella.

Fig. 1.

Location of H-antigen complex secondary antigen target sequences. (a) Conserved EN-complex amino acid sequences targeted by probes to detect single factors H:x and -z₁₅. (b) G-complex amino acid sequences targeted by probes to detect single factors H:f, -m/g,m, -p, and -t.

Fig. 2.

Signal strength of H antigen probes. (a) Raw data averages for five isolates run in the H antigen assay. The signal strength of each H antigen probe is expressed in median fluorescence units, as indicated by the gray bar. Negative-control signal strength is indicated in black on each bar. (b) Average ratios of positive/negative values for five isolates. A positive reaction is represented by a P/N ratio greater than 6.0, as indicated by a dashed line.

Fig. 3.

Frequency of H antigens among the 500 isolates in the evaluation panel.