Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray

Abstract

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

FIG 1

Schematic diagrams of (A) the regions of the carB gene used in the design of the Salmonella positive-control and serotype-specific capture probes and (B) the repeated array format for the carB-based oligonucleotide microarray.

FIG 2

Gel electrophoresis analyses of 18 pathogenic bacteria for PCR amplification of the partial carB gene. Lanes: M, 1-kb DNA size maker; 1, S. Choleraesuis; 2, S. Enteritidis; 3, S. Typhimurium; 4, S. Newport; 5, B. cereus; 6, C. perfringens; 7, L. monocytogenes; 8, S. aureus; 9, C. jejuni; 10, E. coli; 11, S. boydii; 12, S. dysenteriae; 13, S. sonnei; 14, V. cholerae; 15, V. parahaemolyticus; 16, V. vulnificus; 17, Y. enterocolitica.

FIG 3

Detection of three S. enterica subsp. enterica serotypes using the carB-based oligonucleotide microarray. (A) Raw hybridization data with each amplified target at 50°C in hybridization buffer for 1 h and (B) 2D visualization plots of serotype-specific spots. The intensities are represented by gray gradation: the maximum intensity is white, and the minimum intensity is black. (a) S. Choleraesuis; (b) S. Enteritidis; (c) S. Typhimurium. A set of two serotype-specific capture probes was used for S. Choleraesuis (1), S. Enteritidis (2), and S. Typhimurium (3).

FIG 4

Dynamic detection range of the carB-based oligonucleotide microarray. Shown are normalized fluorescence intensity plots for the positive-control (closed circle), specific ST-1 (open circle), and specific ST-2 (closed triangle) spots according to various S. Typhimurium DNA concentrations. The inset is the plot for the specific ST-1 spot at a lower range of DNA concentrations. Each value is the mean of 4 repeated spots, and the error bars represent standard deviations.

FIG 5

Detection of S. Typhimurium from samples of pure DNA (a) and a diverse bacterial-DNA mixture (b). Shown are raw hybridization images (A) and the bar graph (B) of the normalized spot intensities from the serotype-specific spots (ST-1 and ST-2). A set of two serotype-specific capture probes was used for (1) S. Choleraesuis, (2) S. Enteritidis, and (3) S. Typhimurium. Each value is the mean of 4 repeated spots, and the error bars represent standard deviations.