[Examination of Aflatoxin M Contamination in Milk and Milk Drinks Available in Fukuyama City].

To determine the relationship between seasons and regions and aflatoxin M (AFM) contamination of milk distributed in Fukuyama City, we conducted a survey once during the summer and once during the winter between June 2018 and January 2019. We compared the AFM contamination levels in milk drinks available in Fukuyama City during the same period, to provide more about the factors causing AFM contamination. All milk samples examined exhibited AFM contamination levels below the standard AFM contamination level (0.

Sequence-specific detection using a universal probe system based on the formation of a four-way junction structure.

We have developed a novel hybridization detection system using a universal probe based on the formation of a four-way junction (4WJ) structure. This methodology employs a combination of two sequence-specific probes and a universal quenching probe, and the same universal probe can be used for any target gene, allowing cost-effective assays. This 4WJ detection is ideal for extensive parallel identification of nucleic acids such as in multiplex polymerase chain reaction (PCR) systems.

Real-time nucleic acid sequence-based amplification (NASBA) using an adenine-induced quenching probe and an intercalator dye.

Aims: We found that an adenine base caused fluorescence quenching of a fluorescein (FL)-labelled probe in DNA:RNA hybrid sequences, and applied this finding to a nucleic acid sequence-based amplification (NASBA) method.

Methods And Results: The present NASBA method employed a probe containing an FL-modified thymine at its 3' end and ethidium bromide (EtBr) on the basis of a combination of adenine-induced quenching and fluorescence resonance energy transfer (FRET) between the FL donor and EtBr acceptor. This NASBA was used to detect Shiga toxin (STX) stx-specific mRNA in STX-producing Escherichia coli, demonstrating rapid quantification of the target gene with high sensitivity.

Rapid and simple method for serotyping of staphylocoagulase using polystyrene latex particles.

The serotyping of staphylocoagulase is widely used in Japan. However, the conventional immunoassay based on neutralization of the antisera is so laborious and time-consuming that it is not widely used in the other countries. In order to overcome these drawbacks we developed a novel staphylocoagulase serotyping method based on a microplate format using polystyrene latex particles.

Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.

We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously.

Development of rapid coagulase serotyping method by PCR and microplate hybridization.

We developed a novel PCR-based method for coagulase serotyping. Coagulase gene amplicons biotinylated by PCR were identified by microplate hybridization (MPH) using serotype-specific probes. The conventional serotyping method, which is strongly dependent on coagulase activity, may sometimes give a mistaken determination of the serotype, especially in cases where there is high coagulase activity.