Novel Loop-Mediated Isothermal Amplification Method for Detection of the JP2 Clone of Aggregatibacter actinomycetemcomitans in Subgingival Plaque

Abstract

We developed a loop-mediated isothermal amplification method that detects the JP2 clone of Aggregatibacter actinomycetemcomitans, which induces aggressive periodontitis in adolescents of North and West African descents. 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.Aggregatibacter (Actinobacillus) actinomycetemcomitans (11) has long been suspected as an etiological factor in periodontitis in juveniles (17,19). Members of a particular clonal lineage (JP2) of A. actinomycetemcomitans (18) show a unique 530-bp deletion (⌬530) in the promoter region of the ltx operon resulting in enhanced production of leukotoxin, a key virulence factor (1). The JP2 clone shows marked racial tropism as it has been isolated almost exclusively from adolescent periodontitis patients of West and Northwest African descent, including both Africans and Arabs (2,3,4,5,6). Substantial evidence shows that the JP2 clone induces aggressive periodontitis in these populations and constitutes a particularly pathogenic subpopulation of A. actinomycetemcomitans (see reference 6a); reviewed in reference 8).Because the presence of 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 distinguish between the JP2 clone and other genotypes of the species. PCR detection of the ⌬530 deletion has been used to identify the JP2 clone in plaque samples and among isolates and to distinguish it from other A. actinomycetemcomitans strains (13,15,20). However, there is a need for a simple and low-technology method for detection of the JP2 clone. For that purpose, the loop-mediated isothermal amplification (LAMP) method is an attractive alternative to conventional PCR (12).The LAMP reaction runs at a constant temperature, usually around 65°C, and uses four primers that recognize six distinct regions of the target DNA, combined with the Bst DNA polymerase large fragment, which has strand displacement activity and is heat inactivated at 80°C (12). The reaction can be accelerated by inclusion of additional loop primers (10). The resulting amplification products are a complex mixture of stem-loop DNAs with inverted repeats and cauliflower-like structures (12) and induce a white precipitate of magnesium pyrophosphate proportional to the amount of amplified DNA and visible to the naked eye (9). Usually, LAMP primers are designed to specifically recognize a target sequence that is unique to the microorganism in question. A LAMP method targeting the dam gene, which is common to all members of A. actinomycetemcomitans, was reported recently (14). We wanted to develop a method that specifically detects the JP2 clone and took advantage of the specific ⌬530 deletion. The low percentage of GϩC in the sequence around ⌬530 made it impossible to design appropriate LAMP primers in...

The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans: evolutionary aspects, epidemiology and etiological role in aggressive periodontitis

“…The sensitivity for the detection of A. actinomycetemcomitans has increased in recent years as a consequence of a change from traditional cultivation methods, which were mostly used earlier ( Frisken et al ., 1990; Könenen et al , 1992; Kamma et al ., 2000). The DNA‐based methods mostly used today can explain some of the discrepancies in the findings reported in various studies ( Riggio et al ., 1996; Chen & Slots , 1999; Poulsen et al ., 2003; Verner et al ., 2006; Seki et al ., 2008). However, differences in carriage rates, not related to methodology, are found (Table 2).…”

“…Some diagnostic tools, such as conventional and real‐time PCR for use in laboratories, are already available ( Brogan et al ., 1994; Poulsen et al ., 2003; Orrù et al ., 2006). Also a loop‐mediated isothermal amplification method for detection of the JP2 clone of A. actinomycetemcomitans in dental plaque samples has been developed ( Seki et al ., 2008). Microbial diagnosis is a necessary prerequisite for preventive purposes as well as for microbiological monitoring during phases of treatment of diseased individuals.…”

Classification, Identification, and Clinical Significance of Haemophilus and Aggregatibacter Species with Host Specificity for Humans

“…PCR-based detection of Haemophilus and Aggregatibacter in clinical material has focused mainly on rapid diagnosis of H. influenzae meningitis (158-161), improved detection of H. ducreyi (142), and identification of the virulent clone JP2 of A. actinomycetemcomitans (162,163). PCR has also been utilized for detection of H. influenzae DNA in culture-negative middle ear fluids where prior antibiotic therapy has made culturing inconclusive (164) and for improved microbiological surveillance of bacterial meningitis in parts of the world where laboratory facilities for immediate culturing of cerebrospinal fluid samples are not available (165).…”

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

“…Seki et al applied the loop-mediated isothermal amplification (LAMP) method for detecting the JP2 clone in subgingival plaque from Moroccan adolescents and found that 48 of 72 samples (66.7%) were JP2 positive [25]. The LAMP technology was very sensitive for the JP2 clone, and the detection limit was 10 genome copies, whereas that with conventional PCR was 100 copies [25,32,33]. These findings are identical to the sensitivity of qPCR, and the detection rate was similar to our result (54.5%).…”

“…Since this is time consuming, detection using cultures is not suitable for rapid diagnosis. Molecular genetics techniques, which are relatively rapid, including conventional PCR and other molecular-based techniques, have been used to detect the JP2 clone from subgingival plaque [24,25]. However, these are qualitative methods, and no completely quantitative molecular-based detection technique for the JP2 clone has been reported [26].…”