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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 4  »  Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Results.



All samples examined showed the amplicon of the ubiquitous bacterial primers. Only one band of the predicted size was present for each clinical sample. Such results suggested that components of the infected teeth did not significantly inhibit the DNA amplification reaction and indicate that bacteria were present in all cases.
The S. anginosus group was detected in 16.7%, F. nucleatum in 14.3%, and B. forsythus in 7.1% of the abscess samples. No pus sample yielded A. israelii, A. actinomycetemcomitans or fungal species. In general, B. forsythus was found in 20% of the cases (16 of 80), S. anginosus in 12% (6 of 50), F. nucleatum in 10% (6 of 60), A. israelii in 5% (2 of 40), and fungi in 2% (1 of 50). No sample was positive for A. actinomycetemcomitans.
The primers used yielded a single amplicon only with strains of the target species. The primer pair specific for the S. anginosus group amplified reference DNA from both S. anginosus and S. intermedius. No cross-reactivity with non-target species was observed for the test primers. Reference DNA and clinical samples that were positive for the microorganisms tested showed only one band of the predicted size (Fig. 1). Reference DNA from C. albicans , C. glabrata , C. parapsilosis , and C. guilliermondii yielded amplicons of 330 bp, 410 bp, 310 bp, and 360 bp, respectively. The clinical sample positive for fungi showed an amplicon of approximately 380 bp.

Figure 1. Agarose gel electrophoresis of representative PCR results.

Agarose gel electrophoresis of representative PCR results
Lane 1, 100 bp ladder;
Lane 2, root canal sample positive for Bacteroides forsythus;
Lane 3, negative control (without DNA);
Lanes 4 and 5, root canal samples positive for Actinomyces israelii;
Lanes 6 and 7, root canal samples positive for Streptococcus anginosus group;
Lane 8, reference DNA from Actinobacillus actinomycetemcomitans;
Lane 9, reference DNA from Candida albicans;
Lane 10, reference DNA from Candida glabrata;
Lane 11, reference DNA from Candida parapsilosis.

The species B. forsythus, F. nucleatum and S. anginosus , which were detected in symptomatic cases, showed no correlation with symptoms as evaluated by the chisquared test ( P > 0.05) and odds ratio calculation (1.37, 0.58, and 0.6, respectively).