Rapid and specific polymerase Chain reaction-enzyme linked immunosorbent assay for detection of escherichia coli LT Toxin from clinical isolates

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) is the most common agent, which causes diarrhea. ETEC is colonized along the cells and produces enterotoxins leading to diarrhea. Different detection methods have been utilized for detection of ETEC heat Labile Toxin (LT) toxins or respective genes. These methods have disadvantages such as high costs and labor time and limitations in handling many samples simultaneously. Objectives: The aim of this study was detection of LT toxin genes in E. coli clinical strains by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Methods: This experimental study was conducted on Iranian children communities from May to November 2014. Forty stool samples were obtained from laboratories and investigated for heat-labile toxin (LT). Specific primers were designed and the DIG -labeled PCR products were bounded to streptavidin-coated wells of a microtiter plate and detected by anti-DIG-peroxidase conjugate. An internal biotin-labeled probe was designed for LT gene and detected with streptavidin. Sensitivity and specificity of the PCR-ELISA method were determined using Enterobacteria strains. Results: Overall, 7.5% of clinically isolated strains were detected as LT positive. The specificity of PCR-ELISA method was 100%. The detection limit of PCR-ELISA was 1.9 pg of genomic DNA. Conclusions: Results showed that this method is fast and sensitive for diagnosing bacteria. Polymerase chain reaction-ELISA is a suitable substitute for all the above factors because it is a quite sensitive, specific and rapid way for detection of LT toxin gene from ETEC strains.