We describe a new procedure for the PCR amplification of the three short tandem repeat (STR) loci; TH01, CSF1PO and TPOX with small amplicon lengths (the Small TCT ). The accuracy of the Small TCT was verified by typing 100 Japanese samples that had been previously typed using an AmpFlSTR Green I PCR amplification kit (Green I Kit). The results using the Small TCT were consistent with those obtained by the Green I Kit. STR typing using the Small TCT was examined from 36 bloodstain samples that had been left for 1 to 25 years at room temperature and compared to that using the Green I Kit. The Small TCT was superior to the Green I Kit for STR typing especially in the case of bloodstain samples that had aged for more than 8 years.

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This review describes the development of the PCR detection kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in food samples. To develop a detection assay, our research team evaluated the optimization of the pre-enrichment broth, the simple DNA extraction method, and the PCR settings. When this detection protocol was used to detect the above pathogenic bacteria, one cell per 25 g of inoculated sample was detected within 24 h. Moreover, there was excellent agreement between the PCR assay and the conventional culture method. The PCR detection assay system was confirmed to be a reliable and useful method for the rapid screening of food products for foodborne pathogens. The assay system was commercialized as a “[TA10] Pathogenic Bacterial PCR Detection Kit”. When this kit was provided to four different laboratories for an extensive validation study, there were no significant differences in detection sensitivity among the laboratories. The detection kit will be valuable as a screening method for foods contaminated with these pathogens, and it will also be useful for identifying the sources of outbreaks of foodborne illness.

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肺炎球菌の莢膜型を判定する検査法として簡便なスライド凝集法とワクチン型別判定まで可能な

PCR法がある。今回2008年12月から2016年9月の間に愛知医科大学病院の侵襲性肺炎球菌感染症（invasive pneumococcal disease; IPD）患者から分離された肺炎球菌59株を対象にスライド凝集法と PCR法を用いた検討を行った。スライド凝集法を用いて莢膜型を決定した後， PCR法を用いて型別することにより， PCR法の労力の削減および手技の簡易化が可能であった。抗原因子6dが含有されていないスライド凝集法で非凝集であった場合に PCR法で6A/6B/6C/6D型にバンドが認められた場合は，6C型と推定することが可能であった。 PCR法で分類不能な非ワクチン型である6C型をスライド凝集法と PCR法を併用することで推定することが可能であり，今後のワクチン効果の評価や莢膜型の疫学集積に有用である可能性が示唆された。

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キクにおいて経済的に甚大な被害を及ぼすキクわい化ウイロイド (CSVd) とキククロロティックモットルウイロイド (CChMVd) を同時に，かつ簡易に検出する方法を開発した．逆転写反応にそれぞれのウイロイドのプライマーを複数用いた場合，その後の PCR によって多くの非特異バンドが現れウイロイド検定は不可能であった．そこで，CSVd と CChMVd を同時に逆転写する 6 merのプライマー (5′ AAAGGA 3′) を 2 種のウイロイドの塩基配列から設計した．6 mer のプライマーが結合する塩基配列は CSVd (gb: AB006737) では186–191番目と245–250番目，CChMVd では231–236番目であった．6 mer のプライマーを用いて CSVd と CChMVd の同時逆転写を行った場合，その後の PCR で非特異バンドを抑えることができた．また，6 mer のプライマーを用いた場合， PCR による各ウイロイドの検出感度は，それぞれのウイロイドを別々に検出した場合と同等であった．組織を注射針で挿し，針先に付着した組織液を直接テンプレートとし，6 mer のプライマーによる RT 反応を行い， PCR を行ったところ，CSVd と CChMVd の同時検出が可能であった．ここで確立した direct RT-PCR 法はキクにおけるウイロイド検定のコストや手間を削減するものとして有用である．

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組織培養時の体細胞突然変異体の発生における内生的あるいは外生的な原因を特定することを目的とし，セントポーリア（アフリカスミレ）‘タミレス’を用いたバイオアッセイ系を開発した．本品種はピンク色の花弁に青い斑点を持つ品種であるが，これは flavonoid 3′,5′-hydroxylase（F3′5′H）のプロモーター領域に存在するトランスポゾン VGs1（Variation Generator of Saintpaulia 1）の切り出しによる．本品種から不定芽を誘導すると親タイプの他に，多くの青色変異体やキメラ変異体が発生する．これらの親タイプと変異体を識別することができるマルチプレックス PCR を用いて，組織培養変異を引き起こす 4 つの候補，すなわち既に外植体に存在している変異細胞の影響，in vitro あるいは ex vitro といったシューティング環境の違い，外植体の切断，さらに植物ホルモン添加の影響を評価した．まず，葉身断片と雄ずいから発生した全シュートに占める組織培養変異率はそれぞれ 46.6％と 56.5％であり，外植体に存在していた変異細胞率から推定された変異体発生頻度（それぞれ 3.6％と 1.4％）よりも高かった．この結果は外植体に既に存在していた変異細胞は体細胞突然変異体発生の主な原因ではないことを示す．in vitro と ex vitro で育成した親株の葉身を用いて ex vitro でシュートを誘導した場合，体細胞突然変異体の発生はそれぞれ 9.2％と 8.5％であったことから，培養変異の発生には外植体を採取する親株の影響はほとんどないと考えられた．また，in vitro で育成した親株から採取した葉身を in vitro で不定芽誘導した場合の体細胞突然変異体の発生率も 4.9％と低かったことから，in vitro 環境そのものは体細胞突然変異体の発生原因ではないと考えられた．一方，10 × 5 mm に切った葉身断片を外植体として植物ホルモンフリーの培地で不定芽誘導した場合，体細胞突然変異体の発生率は 26.4％であった．さらに，植物ホルモンは非切断葉身および切断葉身の体細胞突然変異体の発生率を上昇させた（それぞれ 39.9％と 46.6％）．セントポーリア‘タミレス’を用いたバイオアッセイは短期間かつ簡便なことから，多くの環境要因をスクリーニングすることを可能とし，変異を回避する新しい組織培養技術の開発に寄与するものと考える．

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A highly sensitive and specific PCR assay has been developed to detect the presence of Escherichia coli O157:H7 from naturally contaminated raw milk samples within 10 h. The primers explored in the assay were targeted against the uidR gene specific for all types of E. coli and the fliCH7 gene specific for the h7 flagellar antigen of E. coli O157:H7. The PCR assay developed was found to be highly specific as it produced PCR products of 152 bp (E. coli specific) and 625 bp (E. coli O157:H7 specific). The assay was tested for its specificity against different serotypes of E. coli as well as other pathogenic strains like Salmonella, Shigella, Klebsiella, Enterobacter, Staphylococcus aureus, Lactobacillus and Lactococcus etc. When this PCR assay was directly applied to 24 raw milk samples collected from different sources, E. coli O157:H7 could be detected in one of the milk samples without 4 h enrichment in CT-SMAC broth and three samples after 4 h enrichment in CT-SMAC broth. However, all the pasteurized milk samples gave a negative signal for this organism.

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We applied transcription in vitro following PCR in order to improve the sensitivity and rapidity of microbial detection in aquatic environment with an oligonucleotide microarray. Transcripts of 16S rRNA gene were fluorescently labeled and hybridized on fabricated oligonucleotide chip. The assay sensitivity was evaluated by detecting cultured bacteria inoculated into natural river water. By using our procedure, the assay was completed more rapidly (6 hr) than conventional oligonucleotide microarray assays (>12 hr), and its sensitivity was improved: detection limit was decreased by one order of magnitude. This method might be useful for monitoring pathogenic bacteria in aquatic environments.

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As application of computer graphics extends, requirement to hardcopy increases, too. And an important requirement to the hardcopy is to display three dimensional images. Hologram is effective as three-dimensional display. So, we tried to make hologram using computer graphics images. And examine if this hologram has enough guality as three dimensional hardcopy of the computer graphics images. hologram can be made from moving images. However, virtual image of the hologram is distorted. Therefore, it is necessary to evaluate the distortion of the hologram on graphic display before making hologram. We made three dimensional graphics system to display curve wire frame pictures and to generate images for pre-evaluation the distortion of the hologram. Then we made hologram from moving images made by this system. We now examine that hologram made by computer graphics images is effective as three dimensional hardcopy and enables to display more free images than hologram made by the real image. Moreover, distortion of the hologram can be preevaluated by this system.

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We developed a polymerase chain reaction (PCR) method using primers specific to the capsular polysaccharide biosynthesis genes (cps) to type Actinobacillus pleuropneumoniae serovars 1, 2, 5, 7, and 15. This PCR method may be useful for the typing of serovar 15, which is recently becoming more prevalent, as well as the most prevalent serovars 1, 2, 5, and 7 in Japan.

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A pneumatic system has several advantages, which are cheapness, lightweight, and reliability to human and environment. These advantages are adapted to some research areas, such as industrial lines, medical and nursing cares, and rehabilitation tools. However, the pneumatic system needs several devices; compressor, air tube, and control valve. This research aim to downsize pneumatic system. In this paper, a new method of pneumatic transmission for multi-pneumatic servo system is proposed. The valve for this system consists of two vibrators supported by springs, which was designed with simple and cheap structure. The working principle of the valve is vibrators resonance from pneumatic transmission and it is possible to work as ON/OFF valves without electric wire. Dynamic simulation was used to confirm the working principle of the resonance driving system. A prototype device confirming the principle was designed and developed based on the simulation. The experiments show that this new control system works very well to control two separated valves through single pneumatic tube.

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Arthrogryposis congenita (AMC) is a syndrome characterized by multiple joint contractures present at birth. It needs a long-term habilitation or performing surgical treatments several times. In this study, the experience in eight cases with AMC was reported.
1) In terms of distribution of contracture, in general joints, 4 of 8 cases belonged to “upper and lower limbs type”, whereas in major joints except hands and feet, most cases were divided into “upper limbs type” and “lower limbs type”.
2) Multiple surgical treatments were performed in 5 of 8 cases.
3) Four of 8 cases had an episode of fracture, mainly in the upper limb.
4) Some cases had abnormal delivery or malformation.
5) Every case showed normal mentality, except for one case with mental retardation.
In AMC, the functional prognosises on ADL were expected to be improving, though long-term treatment and habilitation were needed.

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Ten novel microsatellite markers were developed for the western sand lance, Ammodytes japonicus, which has decreased significantly in many fishing grounds because of overexploitation and deterioration of its habitat. The 10 markers were successfully amplified in two sets of

polymerase chain reactions (PCR). A total of 63 individuals collected in two successive years from a single population were used to assess the characteristics of the 10 markers. The number of alleles per locus ranged from seven to 24 with the observed heterozygosity ranging from 0.397 to 0.921. None of the loci deviated significantly from the Hardy-Weinberg equilibrium, and there was no evidence of linkage disequilibrium between any loci-pairs. Almost all of these novel microsatellite markers were also confirmed to be successfully amplified, not only for the other regional A. japonicus, but also for the closely-related Ammodytes heian and Ammodytes hexapterus. These polymorphic microsatellite markers for PCR will largely improve the throughput of microsatellite DNA analysis, and contribute to the effective genetic monitoring of A. japonicus and other sand lances around Japan.

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近年，カルバペネム耐性腸内細菌科細菌の増加が大きな問題となっている。特に，カルバペネマーゼ産生菌は責任遺伝子がプラスミド上に存在し菌種を超えた伝播をきたすため注意が必要であるが，日常的に実施されている薬剤感受性試験ではカルバペネム系抗菌薬に感性の場合もあり存在が見落とされる危険性がある。本研究では，既報文献を参考に6種類のprimerを選択し，プラスミド性カルバペネマーゼ遺伝子，IMP，VIM，OXA-48-like，NDM，KPC，GESを1チューブで検出可能な PCR法の確立を試みた。既存の3種類のPCR試薬について，IMP型5菌種5株，GES型1菌種1株，VIM型2菌種2株，NDM型1菌種2株，OXA-48-like型1菌種1株，KPC型1菌種2株の計9菌種13株のグラム陰性菌を用いた感度の検討および25菌種30株の上記遺伝子非保有グラム陰性桿菌を用いた特異度の検討を実施した結果，我々の PCR法においてはPlatinum^® PCR Master Mix（Applied Biosystems）の使用が最適と考えられた。本法は上記6種類の遺伝子のスクリーニングが1チューブ内で可能であり効率的，経済的であった。また，DNA抽出から遺伝子確認まで3.5時間と迅速性にも優れており有用性が高いと考えられた。

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The research was focused on the polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65°C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65°C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a PCR profile containing all the expected size amplicons.

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 Body fluid identification from crime scene evidence is crucial to determine the type of crime and to verify the victim’s testimony. We previously developed the multiple body fluid-targeted

RT-PCR assay (MM) for simultaneous discrimination of blood, saliva, semen, vaginal fluid and nasal secretion. That procedure enabled precise and comprehensive identification of forensically relevant body fluids, especially in unknown or mixed biological samples. Alternatively, it is possible to narrow down the body fluid type by creating a case summary and performing a presumptive test for the expected body fluid and followed by a confirmatory test. In the present study, single body fluid-targeted RT-PCR assays (SM) were developed for the individual identification of blood, saliva, semen, or vaginal fluids. The repeatability of SM results was evaluated by using the pooled cDNA of each targeted body fluid and comparing it with the results of MM. Then, the peak height and detectability of each marker were compared between MM and SM on individual mixed and unmixed body-fluid stains. Like MM, the normalized values of SM were well reproduced in repeated electrophoreses. The positivity of the body-fluid markers was also well reproduced in repeated PCR amplifications. The average peak heights of some markers were significantly higher in SM than in MM. The detectability of SPTB, MUC7, and SERPINB13, which showed lower average peak heights among body fluid markers, was improved in SM. Comparable positive detection of other markers that showed sufficient amplification was found in SM and MM. The identification of targeted body fluid was performed based on the criteria we previously proposed, showing highly specific results. In conclusion, SM and MM can be performed individually or simultaneously based on the sample condition or case summary in order to precisely identify body fluid samples in forensic laboratories.

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For identification of three species categorized in Streptococcus anginosus group (SAG), genotyping employing PCR for detection of species-specific genes has been widely used. We wondered whether possession of the specific genes correlated to differentiation of housekeeping genes, with an attempt to obtain more information on genetic background of clinical isolates of SAG strains and to evaluate reliability of the PCR. We therefore determined nucleotide sequences of some housekeeping genes of 52 SAG isolates from healthy volunteer to investigate whether the sequences phylogenetically corresponded to the identification by the PCR. The phylogenetic trees drawn with all of the sequences of 16S rRNA, sodA, groEL and rpoB were found to clearly correlate to species identified by the PCR. Interestingly, phylogenetic tree drawn only with single housekeeping gene often did not match with the species identified by the PCR. Our analysis implies even housekeeping genes can have their sequence varieties as independent manner from presence of species-specific genes in SAG strains, and the choice of housekeeping genes for the phylogenetic analyses may result in failure of the species identification.

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これまでの研究で，我々は果実の甘渋性を支配する AST 遺伝子座に連鎖した分子マーカーを単離しているが，これらのマーカーは‘黒熊’由来の交雑集団に対して有効でなく，育種計画において実用的なマーカーではない．そこで本研究では，育種計画の中で簡便かつ確実に完全甘ガキ個体を選抜するための新しい sequence characterized amplified region（SCAR）マーカーの開発を試みた．完全甘ガキ個体と非完全甘ガキ個体の間で多型を示す新たな restriction fragment length polymorphism（RFLP）マーカーである 5R 領域を‘西村早生’，‘次郎’および‘黒熊’由来交雑後代のゲノムライブラリーから単離して解析した結果，3 つの大きな欠損・挿入変異が ast 連鎖領域と AST 連鎖領域の間で確認された．そのうちの 1 つである Indel-3 の近隣にいくつかのプライマーを設計し，SCAR マーカーとしての有効性を検討したところ，2 つの forward primer（AST-F と PCNA-F）と 1 つの reverse primer（5R3R）の組み合わせで行う PCR が最も有効で確実な方法であることが分かった．AST 対立遺伝子に連鎖する 220 bp の断片は‘黒熊’，‘西村早生’および‘会津身不知’のいずれの品種の後代でも共通に検出されることが示された．この PCR は，カキ育種における最も実用的で応用可能なマーカー選抜法であり，この方法によりカキ育種の進展は飛躍的に向上するものと考えられる．

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Taylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. LAMP worked well without nonspecific reactions, and the analytical sensitivities of LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods.

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Distinguishing between Beryx splendens and Beryx mollis (Actinopterygii: Beryciformes) on the basis of external morphology is difficult, and a reliable method of differentiation must be established. In this study, we developed a

polymerase chain reaction (PCR) method with species-specific primers for distinguishing B. splendens from B. mollis. In total, 146 specimens were collected in the North Pacific Ocean, East China Sea, and Southwest Indian Ocean. Provisionally, 115 specimens were identified on the basis of the number of pyloric caeca. Phylogenetic analysis was also performed by using the 146 partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and sequences from a DNA database. Identification by the number of pyloric caeca was consistent with the result of the COI phylogeny, and we surmised that the use of the COI sequences was effective for the differentiation of the two species. PCR with species-specific primers was subsequently developed on the basis of the partial COI sequences. All of the specimens used in the molecular analyses were successfully identified as B. splendens or B. mollis via electrophoresis of the PCR products amplified using the species-specific PCR primers.

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