Polyphosphate-accumulating bacteria that were previously isolated from activated sludge and exhibited high phosphate removal activity were studied taxonomically and phylogenetically. These organisms were gram-positive, coccus-shaped, aerobic chemoorganotrophs that had a strictly respiratory type of metabolism in which oxygen was a terminal electron acceptor. They accumulated large amounts of polyphosphate under aerobic conditions. The major quinone was menaquinone MK-9(H4). The cell wall peptidoglycan contained LL-diaminopimelic acid. The guanine-plus-cytosine content of the DNA was 67.9 mol%. Our isolates were similar phenotypically and chemotaxonomically to Luteococcus japonicus, which was proposed recently as a new genus and species. However, our isolates differed from L. japonicus in cellular fatty acid composition and some other traits. A phylogenetic analysis based on 16S rRNA sequences showed that our isolate differ from the genus Luteococcus and other genera belonging to the high-G+C-content gram-positive group. Accordingly, we concluded that our strain NM-1T (T = type strain) should be assigned to a new genus and species, for which we propose the name Microlunatus phosphovorus.

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An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

This work demonstrates accurate measurement of the amount of substance concentration of low concentration plasmid DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup. Plasmid DNA is a widely used form of DNA, and its quantity often needs to be accurately determined. This work establishes a reference analytical method for direct quantification of low concentration plasmid DNA prepared as reference standards for polymerase chain reaction-based DNA quantification. The model plasmid DNA pBR322 (4361 bp) was stained with a fluorescent dye and was detected in a flow stream in a micro-fluidic channel with laser-induced fluorescence detection, for which the DNA flow was electro-hydrodynamically focused at the centre of the channel. 200 to 8000 DNA molecules in a ∼1 µL sample volume were counted within 2 min in an ‘exhaustive counting’ manner, which facilitated quantitation without calibration. The sample volume was measured and validated from the close agreement of the results of two independent measurement methods, gravimetric determination of water filling the capillary and graphical estimation of actual cross sectional area of the capillary tubing with the image of calibrated scanning electron microscopy. Within the given concentration range, an excellent measurement linearity (R2 = 0.999) was achieved with appropriate data processing for the correction of the events of double molecules (detection of double molecules opposed to single molecule detection assumed, which occurs due to their coincidental passing of the detection zone). The validity of the proposed method was confirmed from the close agreement with the results of quantitation of enzymatically released nucleotides using capillary electrophoresis.

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We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Quenching probe (QProbe) PCR-based methods for quantifying the abundance of Rhodocyclus-related and actinobacterial polyphosphate-accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR) process are described. PCR primer-QProbe sets specific for the 16S rRNA genes of Rhodocyclus-related PAOs (RPAOs) and actinobacterial PAOs (APAOs) were newly designed, and their specificity tested using database searches, a selection of pure cultures, and sludge DNA. The sets were found to be specific for most of the target sequences. QProbe PCR with the newly designed sets was used to enumerate the 16S rRNA genes of RPAOs and APAOs in a laboratory-scale EBPR sludge. The copy number of 16S rRNA genes of RPAOs was larger than that of APAOs before the phosphorus removal performance of the reactor deteriorated, and the amount of both PAOs decreased in the deterioration period. The approaches developed in this study may become a powerful tool for a high-throughput analysis of PAO abundance in EBPR processes.