Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

Background: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes ( 652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. Methods: The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68%) of whom had various clinical forms of periodontal disease. Results: This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2) cells/ml) for all genotypes and a good correlation factor (R2 = 0.97 - 0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method. Conclusion: A low number of Sardinian patients was found positive for Aa infections in the oral cavity, ( just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60% of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection.