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In Japan, it is mandatory that processed foods containing specific allergenic ingredients be clearly labeled as such. Previously, we developed qualitative real-time PCR methods that enable sensitive and specific detection of wheat, buckwheat, and peanut in foods. The results of interlaboratory validation showed that the performance of these methods meets the criteria of the official Japanese government guidelines. In this study, we evaluated the applicability of qualitative real-time PCR as a confirmation test after an ELISA screening test using 60 samples of commercially available processed foods. The results indicated that the qualitative real-time PCR could detect wheat, buckwheat, and peanut in various foods containing allergens with ELISA values of 10 ppm or higher. Next, we evaluated whether the qualitative real-time PCR could be applied as a primary qualitative test that is faster and less likely to miss the target allergens. To do so, we combined it with a simple DNA extraction kit to utilize this method in a different workflow from the official method. As a result, the identical samples that were positive in the workflow from the official method were judged to be positive for the target allergen. In addition, since it was possible to detect samples with an ELISA value of less than 10 ppm, this study suggests that the qualitative real-time PCR has sufficient sensitivity to be used as a primary qualitative test. [ABSTRACT FROM AUTHOR]

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Riptortus pedestris is a major agricultural pest on leguminous plants in South Korea and Japan. Recent studies have revealed that R. pedestris can form beneficial symbiosis with bacteria belonging to genus Burkholderia acquired from soil newly for every generation. Although their physiological interactions are relatively well-understood, infection rate and abundance of the Burkholderia in overwintering natural populations of R. pedestris remain unknown. Therefore, the objective of this study was to characterize Burkholderia infection ratio and clade composition of overwintering R. pedestris populations as well as prevalence and diversity of the genus Burkholderia in soil by conducting a two-year field survey. From the field survey, we found 29 overwintering R. pedestris adults in forested areas nearby soybean fields. Diagnostic PCR analysis revealed that overall infection rate of the symbiotic Burkholderia was 93.1% from overwintering adults. Among the Burkholderia-infected R. pedestris, 70.4% of individuals harbored unclassified Burkholderia clades whereas 22.2% and 7.4% of R. pedestris harbor stinkbug-associated beneficial and environmental (SBE) group and Burkholderia cepacia and complex (BCC), respectively. All R. pedestris were infected with a single clade of Burkholderia. In soil, 56.2% of soil samples were Burkholderia positive, and unlike R. pedestris, multiple Burkholderia clades were detected from 62.2% of those samples. Clade composition of the genus Burkholderia in the samples with the bacteria was 91.1%, 60.0%, 31.1% and 8.8% for plant-associated beneficial and environment (PBE), BCC, SBE and unclassified clade, respectively. [ABSTRACT FROM AUTHOR]

Summary: The potato rot nematode, Ditylenchus destructor , threatens garlic production in Japan. The objectives of this study were to determine the relationships of D. destructor densities in soil, garlic roots and outer skins of garlic bulbs, and damage to bulbs that rot during storage. Ditylenchus destructor densities were evaluated with the real-time PCR method. There was a significant positive correlation between D. destructor densities in soil at planting and those in the outer skin of garlic bulbs at harvest in 2016, but not in 2017. Ditylenchus destructor densities in outer skins at harvest were consistently low when those in roots at harvest were lower than 80 ind. (0.05 g)−1. No damage to garlic bulbs after storage was observed when D. destructor densities in outer skins were lower than 300 ind. (0.05 g)−1. These results indicate that D. destructor densities in roots and outer skins may be a good indicator to estimate nematode damage to garlic bulbs after storage. [ABSTRACT FROM AUTHOR]

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

To reduce crop damage by Asiatic black bears (Ursus thibetanus), we developed a method to identify individual bears that damaged corn crops based on microsatellite analysis using bear DNA obtained from damaged corn. During summer 2004 in Iwate prefecture. Japan, 99 corn-bite samples were collected, of which 30 (30%) yielded sufficient DNA for 6 complete microsatellite loci. We detected that at least 21 individuals (16 males, 1 female, and 4 of unknown sex) had damaged dent corn in 5 fields. Results enabled individual identification of bears from the samples, but more accurate analysis is needed. Moreover, the sex ratio of nuisance individuals was extremely biased to males compared to that of bears killed through control programs. [ABSTRACT FROM AUTHOR]

Background: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible., Methods: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2., Results: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing., Conclusion: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)