ABSTRACT
Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.
Subject(s)
Adult , Aged , Child , Female , Humans , Male , Middle Aged , /chemistry , Shiga Toxin 1/isolation & purification , /isolation & purification , Shigella dysenteriae/chemistry , DNA, Bacterial/genetics , Enzyme-Linked Immunosorbent Assay , /genetics , Feces/microbiology , Genes, Bacterial/genetics , Polymerase Chain Reaction , Sensitivity and Specificity , Shiga Toxin 1/genetics , /genetics , Shigella dysenteriae/geneticsABSTRACT
OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT) and colonization factors (CFs) are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells. METHODS: In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated. RESULTS: Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells. DISCUSSION: The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity. .