) PCR-EL1SA( با استفاده از روش واكنش زنجيره اي پليمر ار اليزا O1 تشخيص سريع ويبريوكلرا

Rapid Detection of Toxigenic Vibrio Cholera 01 Using PCR- Enzyme-Linked Immunosorbent Assay (PCR-ELISA)

Objective: Vibrio cholera 01 is the causative agent of a severe secretary diarrhea and has historically been considered the cause of cholera epidemics. Recent epidemics of cholera in various parts of the world have emphasized the urgent need for rapid and reliable methods for detection of Vibrio cholera 01. Classical and conventional microbiological techniques that are sensitive and specific require several days to complete. Therefore, in this study a new technique for detection of vibrio cholera 01, using PCR-Enzyme-linked Immunosorbent assay (PCR-ELISA) was developed. Material and Methods: After amplification of a 375-bp sequence of a gene that codes for the synthesis of cholera toxin B subunit, the digoxigenin-labeled amplified product was captured on PBS buffer, coated on micro titer plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The specificity of the PCR was determined using 5 strains of others bacteria including salmonella paratyphoid C , Klebsiella , shigella dyscentriae, Enterobacter and Pseudomonas aeruginosa. The PCR product obtained was hybridized with biotin labeled probe and ELISA using streptoavidin observed the result. Result : The sensitivity of the PCR-ELISA test for genomic DNA as a template was 0.5 -0.6pg of DNA whereas it was 5 cells when lyses bacteria were used as a DNA source. Conclusion: The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and that obtained by conventional PCR assay. The PCRELISA technique seems to be a practical and reliable tool for the diagnosis of vibrio cholera 01.