PCR detection of Salmonella enterica serotype Montevideo in and on raw tomatoes using primers derived from hilA

Abstract

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from hilA and sirA, positive regulators of Salmonella invasive genes, were developed to identify Salmonella enterica serotype Montevideo on and in tomatoes. Based on examination of 83 Salmonella strains and 22 non-Salmonella strains, we concluded that a pair of hilA primers detects Salmonella specifically. The detection limits of the PCR assay were 10(1) and 10(0) CFU/ml after enrichment at 37 degrees C for 6 and 9 h, respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated tomatoes, 10(2) and 10(1) CFU/g were detected, respectively, after enrichment for 6 h at 37 degrees C. Our results suggest that the hilA-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.

FIG. 1

Specificity of HILA2-based PCR assay for Salmonella: gel electrophoresis of PCR products in 1% agarose in 1× TBE buffer. (A) Salmonella strains. Lanes 1 and 22, 100-bp DNA ladders (GIBCO BRL); lanes 2 through 20, PCR products amplified from S. enterica serotype Anatum, S. enterica serotype Baildon, S. enterica serotype Cubana, S. enterica serotype Enteritidis, S. enterica serotype Gaminara, S. enterica serotype Hartford, S. enterica serotype Heidelberg, S. enterica serotype Infantis, S. enterica serotype Michigan, S. enterica serotype Montevideo, S. enterica serotype Muenchen, S. enterica serotype Newport, S. enterica serotype Oranienburg, S. enterica serotype Panama, S. enterica serotype Poona, S. enterica serotype Saintpaul, S. enterica serotype Thompson, S. enterica serotype Typhimurium, and S. enterica serotype Typhimurium DT 104, respectively; Lane 21, negative control. (B) Non-Salmonella strains. Lanes 1 and 22, 100-bp DNA ladders (GIBCO BRL); lane 2, S. enterica serotype Typhimurium; lanes 3 through 20, the negative results obtained with Aeromonas sobria (lane 3), Escherichia coli ATCC 10789 (lane 4), Escherichia coli O157:H7 (lanes 5 and 6), Enterobacter aerogenes (lane 7), Klebsiella pneumoniae (lane 8), Serratia marcescens (lane 9), Shigella dysenteriae non-type I (lane 10), Shigella sonnei, (lanes 11 through 13), Staphylococcus aureus, (lanes 14 and 15), Proteus vulgaris (lane 16), Pseudomonas fluorescens (lane 17), Yersinia enterocolitica (lanes 18 through 20); lane 21, negative control.

FIG. 2

Limits of detection of HILA2-based PCR assay. (A) PCR products amplified from crude DNA. Lane 1, 100-bp DNA ladders; lanes 2 through 10, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/ml, respectively; lane 11, negative control. (B) PCR products amplified from purified DNA. Lane 1, 100-bp DNA ladders; lanes 2 through 10, DNA equivalent to 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/ml, respectively; lane 11, negative control.

FIG. 3

Limits of detection of HILA2-based PCR assay with different enrichment times. Cell cultures containing 10⁹ CFU/ml were serially diluted to concentrations of 10⁸ to 10⁰ CFU/ml, and 100-μl portions of the dilutions were transferred to 900-μl portions of BHI broth, which were then incubated at 37°C with shaking for 3, 6 and 9 h. The cells were treated, and DNA was amplified by PCR. Lanes 1 and 21, 100-bp DNA ladders; lanes 2 through 7, PCR products from preparations containing 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/ml, respectively, after 3 h of enrichment; lanes 8 through 13, PCR products from preparations containing 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/ml respectively, after 6 h of enrichment; lanes 14 through 19, PCR products from preparations containing 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/ml, respectively, after 9 h of enrichment; Lane 20, negative control.

FIG. 4

Use of HILA2-based PCR assay to detect S. enterica serotype Montevideo on and in artificially inoculated tomatoes. Tomatoes were inoculated with different levels of S. enterica serotype Montevideo, and cells were then recovered and enriched for 6 h. Crude DNA was prepared and subjected to PCR amplification. (A) Detection of S. enterica serotype Montevideo in tomatoes. (B) Detection of S. enterica serotype Montevideo on tomato surfaces. Lane 1, 100-bp DNA ladders; lanes 2 to 7, PCR products amplified from preparations containing 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU/tomato, respectively; lane 8, negative control.