Detection of Shigella dysenteriae STX1 gene from Mazandaran province clinical samples by PCR-ELISA method

Abstract

Background:Among enterobacteriaceae bacteria, Shigella dysenteriae produce a shiga protein toxin, and have cytotoxicity, enterotoxin and neurotoxin activity. The toxin causes diseases such as diarrhea, gastroenteritis, intravascular coagulation disorder. Today diarrhea is the most important challenge for the human health. The classic and conventional microbiological detection methods are sensitive and specificity, there is limitation. This study was designed to identify shigella dysenteriae in shortest time and the amount of toxin Stx1 with high sensitivity and specificity by using PCR-ELISA method. Method and material: The Stx1 sequence (490bp) as a target gene was amplified by Dig-dUTP labeled, then product was coating on microplate and by using anti antibody digoxigenin conjugated detection was done. Also the specificity and sensitivity of method with clinical specimens examined. Results: Sensitivity detection of bacteria in the sample using genomic DNA for Shigella dysenteriae was 1/56pg and specificity of technique didn’t showed acceptable OD in other species of enterobacteriaceae family. ELISA for genomic DNA Up to dilution 0/156 pg had significant absorption. As well as from 70 clinical samples which was analyzed, 3 samples contained stx1 gene. Conclusion: Efficiency PCR-ELISA techniques showed that it was simple, faster, high specific and sensitive methods. This technique is more suitable than culture and PCR method also it was easily can applied in each medical laboratory.

Keywords

• Shigella dysenteriae
• Shiga toxin
• Digoxigenin
• PCR-ELISA