DETECTION OF BACTERIA BY AMPLIFYING THE 16S rRNA GENE WITH UNIVERSAL PRIMERS AND RFLP

Background: There is a conserved portion in the 16S rRNA gene of bacteria which can be amplified by the universal PCR method. This fragment is 996 bp in length. In this method, only one set of universal primers is used for the amplification of the conserved region of the 16S rRNA gene, in common bacterial pathogens. Therefore, using the universal PCR method, these bacteria are detectable only by one set of primers then for detection of the bacteria, the PCR products are digested by the restriction endonucleases. Since the restriction patterns of bacteria (RFLP) are expected to be different from each other, on that basis we can identify the bacteria. Methods: The conserved fragments of the 16S rRNA genes ofthe following bacteria were amplified by the universal PCR method: Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa and Neisseria gonorrhoeae. The PCR products were digested by BsuRI (Hae III) restriction endonucleases and were electrophoresed on agarose gel. Results: The restriction patterns of these bacteria were different. Thirty isolated E. coli and 28 isolated S. pyogenes from clinical samples were studied by this method. The size of PCR products and RFLP patterns of every bacterium were the same as standard strains. In comparison with culture method, the sensitivity of the universal PCR is 92.3 %.The sensitivity of this method was determined up to about 11 and 190 bacteria for gram negatives and gram positives respectively. Conclusion: These studies suggest that the universal PCR method accompanied with RFLP is a very useful and rapid method, for detection and identification of bacteria in body fluids.