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.


CONCLUSIONS
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.
Orrù
Germano
G
O.B.L. (Oral Biotechnology Laboratory), Dipartimento di Chirurgia e Odontostomatologia Università degli Studi di Cagliari, Cagliari, Italy. orru@unica.it
Marini
Mario Francesco
MF
Ciusa
Maria Laura
ML
Isola
Daniela
D
Cotti
Marina
M
Baldoni
Marco
M
Piras
Vincenzo
V
Pisano
Elisabetta
E
Montaldo
Caterina
C
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016976:Aggregatibacter actinomycetemcomitans / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D052160:Benzothiazoles; D002648:Child; D003773:Dental Plaque / Q000382:microbiology*; D003959:Diamines; D005098:Exotoxins / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D009930:Organic Chemicals / Q000737:chemistry; D010510:Periodontal Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012463:Saliva / Q000382:microbiology*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005098:Exotoxins; D009930:Organic Chemicals; D011804:Quinolines; C018579:leukotoxin; C098022:SYBR Green I