[Development of a PCR Method to Detect a Mysid Species Causing False Positives in Tests for Crustacean Allergens].

Mysids are small crustaceans that are closely related to shrimp/prawns and crabs but not subject to food allergen labeling requirements for raw materials. In the past, a processed food that contained Japanese smelt (wakasagi) was suspected of producing a false-positive result in shrimp/prawn and crab allergen test because of the presence of consumed mysids. However, there was no reported methods to confirm mysid presence. Therefore, we developed a PCR method to detect mysids. The developed PCR method had high specificity for a mysid species, with no amplification observed from samples of shrimp, crab, krill, mantis shrimp, or the meat of Japanese smelt. In addition, DNA extracted from the internal organs of Japanese smelt was amplified by this PCR method, and sequencing revealed mysid DNA. This confirmed that mysids remained in the internal organs of Japanese smelt following consumption. This PCR method for mysid detection even amplified Japanese smelt-containing processed food samples that were suspected to have produced a false-positive result in shrimp/prawn and crab ELISA. Thus, this PCR method would enable to detect such false positives are caused by mysid contamination.

[Verification Study of the Detection Method for Unauthorized Genetically Modified Papaya by Combining DNA Polymerases and Real-time PCR Instruments].

In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.

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.