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. 2010 Oct;41(4):993-1000.
doi: 10.1590/S1517-838220100004000018. Epub 2010 Dec 1.

Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

Kenia Barrantes Jiménez  1 , Clyde B McCoy, Rosario Achí
Affiliations

Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

Kenia Barrantes Jiménez et al. Braz J Microbiol. 2010 Oct.

Abstract

A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 10(7) CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 10(4)CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 10(6) CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture.

Keywords: PCR; Shigella; food; lettuce; rapid method.

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Figures

Figure 1
Figure 1
Multiplex PCR standardization results: 2% agarose gel electrophoresis stained with ethidium bromide. Lane 1: molecular size marker; lane 2: negative control, lane 3 and 4: Shigella flexneri strain 50(ipaH+/ial+); lanes 5 and 6: Shigella flexneri strain 01 (ipaH+/ial+).
Figure 3
Figure 3
Multiplex PCR results from direct-PCR samples at different concentrations of Shigella flexneri. 2% agarose gel electrophoresis stained with ethidium bromide. Lane 1: empty; lane 2: molecular size marker; lane 3: 107 Shigella direct-PCR sample, lane 4: 106Shigella direct –PCR sample; lane 5: 105Shigella direct-PCR sample; lanes 6 and 9: negative control; lanes 7 and 11, 105Shigella enriched sample; lane 8 and 12: 106Shigella enriched sample; lane 10: Escherichia coli.
Figure 2
Figure 2
Multiplex PCR results from Enriched-PCR samples at different concentrations of Shigella flexneri. 2% agarose gel electrophoresis stained with ethidium bromide. Lane 1: negative control, lane 2: molecular size marker; lane 3: Escherichia coli; lane 4: Shigella 106 enriched-PCR sample; lane 5: Shigella 105 enriched-PCR sample; lane 6: Shigella 104 enriched-PCR sample; lanes 7 and 8: empty.
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