Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 g/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.

Three new compounds, enhygromic acid (1) and deoxyenhygrolides A (2) and B (3), were isolated from a marine myxobacterium, Enhygromyxa sp. Compound 1 was found to be an acrylic acid derivative with a rare polycyclic carbon skeleton, decahydroacenaphthylene, by spectroscopic analyses. Compounds 2 and 3 were deoxy analogs of the known γ-alkylidenebutenolides, enhygrolides. Compound 1 exhibited cytotoxicity against B16 melanoma cells and anti-bacterial activity against Bacillus subtilis, and enhanced the NGF-induced neurite outgrowth of PC12 cells.

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.A real-time PCR assay has been developed for quantifying DNA. This method allows easy, rapid, quantitative detection of microorganisms in clinical samples (7,21). Quantitative analysis with the identification of periodontopathic bacteria is important for the diagnosis, therapeutic evaluation, and risk assessment of periodontal disease (8,14,17,18,20,22).Of the periodontopathic bacteria, Prevotella species form a major portion of the microflora found in human gingival crevices in patients with periodontal disease (10). Prevotella intermedia and Prevotella nigrescens were recently distinguished from each other (19). The two previously recognized genotypes of P. intermedia were elevated to species rank as P. intermedia, corresponding to genotype I, and P. nigrescens, corresponding to genotype II. (6). Thus, identification of the species-specific region between genotypically similar species is essential for the detection of closely related strains, especially in oral biofilms. However, these organisms share over 94% identity on the basis of their 16S rRNA sequences (12). Furthermore, whole-genome sequence information for P. nigrescens is not yet available. Therefore, we used the genomic subtractive hybridization technique to identify species-specific regions that distinguish P. intermedia and P. nigrescens. This technique has successfully identified genomic differences between closely related strains (1). This report first describes the identification of a speciesspecific sequence for P. nigrescens using subtractive hybridization, the design of a P. nigrescens primer-probe set using this technique, and the quantification of four representative oral black-pigmented Prevotella species using a TaqMan real-time PCR assay.The bacterial strains used in this study are listed in Table 1. The P. intermedia, P. nigrescens, Prevotella melaninogenica, and Prevotella loescheii strains were cultured anaerobically (10% CO 2 , 10% H 2 , 80% N 2 ) at 37°C in GAM broth (Nissui Medical Co., Tokyo, Japan) supplemented with hemin (5 g/ml) and menadione (0.5 g/ml). The isolation of genomic DNA and treatment of subgingival plaque samples from patients were performed as previously described (16,21). Briefly, genomic DNA was isolated and purified using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) in accordance with the manufacturer's instructions for gram-negative bacte-

Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.

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