Rapid DNA template preparation directly from a rice sample without purification for loop-mediated isothermal amplification (LAMP) of rice genes.

Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris-HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.

[Qualitative Real-Time PCR Method for Poisonous Entoloma rhodopolium-Related Species in Japan: Real-Time PCR Method for Entoloma Mushrooms].

Qualitative real-time PCR method for three poisonous Entoloma rhodopolium-related species in Japan was established using specific primers and FAM, VIC, Texas Red, Cy5-labeled probes. The use of multicolor probes can extend the method to simultaneous detection of different targets. Standard plasmids were constructed as reference materials. Designed primers and probes in the method detect only a target species, and the detection limit was 12.5 copies or below. This indicates it is highly specific and sensitive enough to detect the poisonous mushrooms in food residues. Next, we applied the method to four food residue samples obtained from food poisoning cases. The real-time PCR method did identify all of four samples as E. subrhodopolium and E. pseudorhodopolium, whereas PCR-RFLP did not. The method established here revealed Entoloma rhodopolium-related species in Hokkaido were different species such as E. eminens and unknown species.

Loop-mediated isothermal amplification (LAMP) for rapid and easy identification of Omphalotus japonicus.

Omphalotus japonicus is a major toxic mushroom in Japan. When food poisoning caused by O. japonicus occurs, quick and accurate identification using a method that does not rely on morphological discrimination is required. Because the loop-mediated isothermal amplification (LAMP) method meets these requirements, we developed a LAMP method for detecting O. japonicus. Amplification occurred within 60 min, and the presence or absence of O. japonicus was confirmed within 2 h, including the DNA extraction protocol. The LAMP method did not show cross-reactivity with 13 species of edible mushrooms, had high specificity toward O. japonicus, and had sufficient detection sensitivity even in a mixed mushroom sample containing 1% O. japonicus. Additionally, O. japonicus could be detected in simulated food poisoning samples of heated and digested mushrooms, and in actual food poisoning residual samples.

Introduction of amino acid residues at the N-terminus of the zeocin-resistance protein increases its expression in Saccharomyces cerevisiae.

Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.e. ATG at the position -9 through -7, which resulted in the incorporation of three amino acids at the N-terminus of the protein. The introduction of these sequences upstream of the initiation codon increased the expression levels of zeocin-resistance protein by 2.2-6.5-fold. One of these sequences increased the expression levels of three out of four human proteins, thereby suggesting that this sequence may also enhance the expression efficiency of mammalian proteins in yeast.

Rapid Identification Method of Omphalotus japonicus by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).

Omphalotus japonicus is a poisonous mushroom that grows in Japan. It can be mistaken for edible mushrooms (Shiitake, Hiratake and Mukitake), and if ingested, it causes food poisoning within 30 min to 1 hr. We established a rapid detection method using PCR-RFLP to identify O. japonicus by restriction digestion of the amplified ITS region. By using Sau96I, Bpu10I, SfcI or DrdI/HincII as a restriction enzyme, it was possible to rapidly identify and discriminate O. japonicus based on the fragment length. This study also provided a short PCR-RFLP system comprising amplification and digestion of a short 200-bp DNA fragment within the ITS region. The system could identify and discriminate O. japonicus after in vitro gastric digestion of native and heated mushroom samples as a model of food poisoning. In addition, a confirmatory assay using real-time PCR was developed to achieve more sensitive detection of O. japonicus.