A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products.

The onset of plaque-mediated disease, including dental caries and periodontal diseases, is highly associated with compositional change of the resident microflora from the ecological perspective. As specific bacterial profiles have been linked to different disease stages, microbial compositional measurements might therefore have great value for clinical diagnosis. Previously we have reported a dry-reagent strip biosensor-PCR-dipstick DNA chromatography, which utilized molecular recognition of oligonucleotides and biotin-streptavidin, and the optical property of colored microspheres, for semiquantifying a five-membered subgroup of caries-associated bacterial species in supragingival plaque from healthy coronal surfaces of teeth. The present study aimed to evaluate this technique's ability to differentiate microflora by comparing the subset profiles. Sixteen subgingival plaque specimens were pooled from periodontal pockets and analyzed for the composition of Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces sp. and Veillonella parvula. Detection frequencies, relative abundance of each bacterial species, and the five-membered bacterial profiles were compared between supra-and subgingival groups. The supragingival plaque harbored significantly more of the tested species and higher amount of Actinomyces sp. and V. parvula. In subgingival plaque, the predominance was obscured, since several highly overlapped profiles were found at comparable frequencies. Thus, PCR-dipstick DNA chromatography using the same plaque sample enabled simultaneous profiling of multiple species at species level and facilitated discrimination between anticipated different microflora, making this technique a promising chair-side microbiota profiling method.The ecological plaque hypothesis (18) has revolutionized our view of the etiology of the most prevalent oral diseases, including caries and periodontal diseases. Contrary to the classical medical model, which ascribes an infection to a single microbial pathogene, the new hypothesis suggests that a dysbiosis of the resident oral microflora, driven by an ecological perturbation, leads to plaque-mediated disease. During the transition from oral health to disease state, a dynamic interaction between the resident microflora and the habitat has been evidenced, with numerous bacterial species being implicated (2,3,22,31). A substantial change of the environmental determinants could disrupt the homeostasis of the resident microflora, thereby inducing the prolifera-

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.