RAHMANI S, FOROZANDEH M, MOSAVI M, REZAEE A. DETECTION OF BACTERIA BY AMPLIFYING THE 16S rRNA GENE WITH UNIVERSAL PRIMERS AND RFLP. Med J Islam Repub Iran 2006; 19 (4) :333-338
URL:
http://mjiri.iums.ac.ir/article-1-560-en.html
Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modarres University , foroz@modares.ac .ir
Abstract: (7872 Views)
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.