Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.
The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.
García
L
L
Departamento de Bioquímica, Facultad de Medicina, Universidad Complutense de Madrid, Spain.
Tercero
J C
JC
Legido
B
B
Ramos
J A
JA
Alemany
J
J
Sanz
M
M
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000235:genetics; D003773:Dental Plaque / Q000382:microbiology; D005784:Gene Amplification; D016680:Genome, Bacterial; D006801:Humans; D010518:Periodontitis / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000302:isolation & purification*; D018713:Prevotella intermedia / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012336:RNA, Ribosomal, 16S