Specific primer for AP-PCR identification of Actinobacillus actinomycetemcomitans.
We used arbitrarily-primed polymerase chain reaction (AP-PCR) to design and construct a specific primer pair for the identification of Actinobacillus actinomycetemcomitans. We analyzed 25 DNA samples of A. actinomycetemcomitans isolated from patients with localized-juvenile periodontitis. From 90 AP-PCR primers screened, one amplification product was selected, cloned in pCR II vector, and sequenced. The sequence was used to design a single pair of specific primers. The sequence was compared with GenBank entries using BLAST and showed no significant matches. PCR amplification using the new primer pair AA1416 produced a characteristic 3.5-Kb band in all A. actinomycetemcomitans DNAs tested. Primer pair AA16S produced no or different amplicon profiles using DNA samples from bacterial species other than A. actinomycetemcomitans. Our results show that this single primer pair AA1416 can be used in PCR to identify A. actinomycetemcomitans isolates and differentiate them from other periodontal bacteria. These approaches appear promising in facilitating laboratory identification and taxonomy of putative periodontopathogens.
Avila-Campos
M J
MJ
Department of Microbiology, University of São Paulo, SP, Brazil. mariojac@icb.usp.br
Sacchi
C T
CT
Whitney
A M
AM
Steigerwalt
A G
AG
Mayer
L W
LW
D016976:Aggregatibacter actinomycetemcomitans / Q000145:classification* / Q000235:genetics; D010520:Aggressive Periodontitis / Q000382:microbiology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S