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

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.

Microbiological Quality of Leafy Green Vegetables Grown in the Toluca Valley

“…However, there are studies highlighting that the O antigen gene cluster of E. coli O105 has the same genes and organization from Shigella boydii O11, which may influence the survival and invasion of the bacteria, which are presented as a risk factor, even when it is thought that washing can eliminate this type of contamination (Tao et al, 2004;Kniret et al, 2016). Likewise, several studies have identified faecal contamination and the presence of pathogenic microorganisms in vegetables such as spinach, lettuce and other crops for fresh consumption (Jiménez et al, 2010;Puig et al, 2014). In some cases, they did not exceed the maximum permissible limit; however, there were enteropathogenic microorganisms dangerous to humans.…”

Advanced Studies on Virulence Genes of Salmonella and Shigella species Isolated from Milk and Dairy Products

“…The PCR products were electrophoresed on a 1 % agarose gel at 100 V. The agarose gel was stained with 0.5 μg/ml ethidium bromide. The DNA band was visualised by gel documentation system (Biorad, USA) [16,21,22,23].…”