PCR-dipstick DNA chromatography for profiling of a subgroup of caries-associated bacterial species in plaque from healthy coronal surfaces and periodontal pockets.

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.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

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.

Tag/hybridization-based sensitive detection of polymerase chain reaction products.

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

A rapid and enhanced DNA detection method for crop cultivar discrimination.

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.