Detection of the virulence plasmid pINV, using <I>inv</I>E gene in <I>shigella</I> sp. in sewage samples by PCR assay.

Abstract

Virulent phenotype of Shigella is directly related to the presence of pINV plasmid of 210 to 230 kb. IpaBCD proteins encoded by the plasmid direct the bacterial invasion into the host epithelial cells. InvE protein positively regulates the expression of virulence genes ipaBCD. The polymerase chain reaction was used to amplify a 362 base-pair fragment specific for the invE gene in sewage samples. Sewage samples were used in the assays because invasive bacteria like Shigella can used this way to infect human. The amplification products were digested with the restriction enzyme BglI and the resulting fragments confirmed the specificity of the amplification. A detection limit of 105 CFU/mL of Shigella was obtained for cultured bacteria and also for sewage samples seeded with the pathogen. Sewage samples were seeded with S. dysenteriae in enrichment culture and incubated for 8h at 37ºC. After incubation DNA was extracted and the PCR assay ensued. A minimal concentration of 102 CFU/mL was enough for the detection of the microorganisms after the enrichment of the sample. Key words: PCR, sewage, Shigella sp., invE gene, enrichment culture.