Novel LAMP Method Detecting the JP2 Clone of Aggregatibacter actinomycetemcomitans

A particular genotype, termed the JP2 clone, of Aggregatibacter actinomycetemcomitans constitutes a unique pathogenic subpopulation of the species that causes aggressive periodontitis in juveniles of African descent. One of the characteristics of the JP2 clone is a 530 bp deletion in the promoter of the leukotoxin gene operon resulting in enhanced production of leukotoxin. Because infection with the JP2 clone has significant implications for prevention and treatment of periodontitis in juveniles it is essential to be able to accurately determine its presence in subgingival plaque samples. Cultivation by itself does not identify the JP2 clone. PCR detection has been used for this purpose; however, a simple and low-technology method for detection of the JP2 clone is necessary. The LAMP method is an attractive alternative to conventional PCR.

Objectives: To develop a LAMP method for detection of the JP2 clone of A. actinomycetemcomitans in subgingival plaque samples. Methods: Because of the low GC percentage in the sequence around the 530 deletion, it was impossible to design appropriate LAMP primers in this region including a primer spanning the site of deletion as usual. As an alternative, we developed a novel approach and designed LAMP primers based on the difference in distance between primer regions (“spacer regions”) in the JP2 clone and in other A. actinomycetemcomitans.

Results: The reduced distance between primer regions increases the amplification efficiency and results in an amplification product only from A. actinomycetemcomitans bacteria of the JP2 clone. The method is highly specific and the detection limit is 10 genome copies. Conclusion: The LAMP method detects only the JP2 clone of A. actinomycetemcomitans in plaque samples. Being independent of special equipment this specific and sensitive method offers significant advantages for screening of patients on a population basis and in clinical settings.