In situ polymerase chain reaction (ISPCR)の方法,応用,問題点について検討した. In は目的とするDNAを組織, cytospin, またはcell suspensionのなかで増幅させる方法であり,感度を上げるだけでなく,その局在がわかるという利点がある.しかし,非特異的反応という問題点がある. Direct ISPCRは,目的とする核酸に直接標識した核酸を取り込ませていくものであるが, DNAおよびTaq DNA polymerase依存性でprimer非依存性の非特異的反応が認められる. Direct ISPCRでは, no primerまたはirrelevant primerによるコントロールが必要である. Indirect ISPCRは, PCRとin situ hybridization (ISH)を組み合わせたものであるが, Taq DNA polymeraseをPCRのステップで除くコントロールとISHの条件をきちんと設定することが重要である. Reverse ISPCRは, reverse transcriptaseを用いて, mRNAからcDNAをin situで作製しPCRを行うものであるが, DNase処理のコントロールとirrelevant primerのコントロールが必要である. In の結果を記載するときには,非特異的反応の可能性を除くために,コントロールの結果が記載されるべきである.
Direct ISPCRで認められるこの非特異的反応の原因は,まだはっきりしていない.われわれは,これを解決するためにdideoxy dNTPs, DNA ligase, nuclease S 1, T 7 exonucleaseによる前処理やPCRにおいてTaq DNA polymerase Stoffel fragmentを用いる処理を単独または組み合わせて行ったが,解決できなかった.したがって,非特異性の機序として,目的とする核酸にnickがあるためという可能性や組織中にあるDNAの小断片がprimerのように働いて生じるという可能性以外の原因を考える必要がある.
In は非常に感度のよい方法なので,多くのことに応用が可能であると思われる.特に臨床材料から病原微生物を検出することはコントロールもおきやすく,よい適応と思われる.しかしながら,その実施に当たっては,非特異性の除去のために十分なコントロールがおかれるべきである.

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The most innovative technique introduced recently into the field of biological study is the polymerase chain reaction (PCR) which can amplify even single-copy DNA sequences to high levels. In , which is a method of in Situ amplification of DNA of interest in tissue sections, has been subsequently developed and mainly used to investigate tissues infected with viruses. A combination of reverse transcription and PCR has been utilized to detect mRNA of interest in Situ. The technique of in is promising for a wide variety of applications in basic histology and histopathology.

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Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in , in /hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10¹ to 10⁵ 50% tissue culture infected doses (TCID₅₀), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in , PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions.

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Direct in 法は特定のDNA配列を標的として菌体内でPCRを行い, 増幅されたDNAを蛍光基質などでラベルすることにより, 蛍光顕微鏡下で特定遺伝子を持つ細菌を単個細胞レベルで特異的に検出する方法である。PCRには腸管出血性大腸菌の Vero 毒素をコードする遺伝子 slt-I, slt-II に特異的なEVT, EVSプライマーを用いた。増幅されたDNAはジゴキシゲニンにより標識し, アルカリホスファターゼ標識した抗ジゴキシゲニン抗体および蛍光基質としてHNPP/Fast Red TRを用いた。その結果, E. coli O157を蛍光顕微鏡下で特異的にまた高感度に単個細胞レベルで検出することができた。

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Human papilloma virus (HPV) infection has been implicated in the etiology of oral cancer. The frequency of detection of HPV in oral cancer tissues ranges from 0 to 100%. Some of this variability can be attributed to the various methods of detection. Therefore, we developed a highly sensitive HPV detection system using the in situ polymerase chain reaction (PCR). It makes use of the extreme sensitivity of PCR and the localizing ability of in situ hybridization. The HPV primers (MY 09 and MY 11) used in this study target the highly conserved L1 gene coding for the major component of viral capsid protein, and amplify 450 base-pair product from more than 50 HPV types. A number of control experiments were performed to prevent false positive reactions and to find the optimal conditions. In appears to be an effective technique for examining HPV in oral lesions.

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Although human papillomavirus (HPV) infection has been shown to be a significant carcinogen in cervical squamous cell carcinoma (SCC), its significance in oral SCC remains unclear. We developed highly sensitive detection methods for HPV to elucidate the prevalence and localization of HPV in paraffin sections from human oral SCC using modified in and in situ hybridization AT tailing (ISH-AT). Analyses revealed a high prevalence of several HPV types (HPV-16, 18, 22, 38 and 70) under optimal conditions. The ISH-AT method can be used as an alternative to in . Various staining patterns were observed in the 20 cases and HPV-positive cells were localized within the surface epithelium as well as in neoplastic cells. We demonstrated that HPV DNA could be detected in paraffin sections using either the method of in or ISH, providing an appropriate primer and probe are used.

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We performed two independent evaluations of in situ polymerase chain reaction (in ) and Epstein-Barr virus encoded small RNA in situ hybridization (EBER ISH) for detection of Epstein-Barr virus infection in gastric carcinoma. In is a novel technique with a high sensitivity which is a powerful aid for morphological diagnosis. Paraffin sections of gastric carcinomas from 96 cases were analyzed. Twenty-two of 96 cases (23%) were positive by in , and 14 cases (15%) were positive by EBER ISH. Therefore there were 8 cases of EBV-DNA-positive gastric carcinoma without expression of EBER. To determine the significance of the expression of EBER in EBV-infection positive gastric carcinoma, we performed a clinicopathological study. There were two pathologic features in the gastric carcinoma with EBER. There was a significantly higher incidence of undifferentiated type and advanced cancer in EBER-positive gastric carcinomas. Therefore the expression of EBER contributes to the clinical pathologic feature of Epstein-Barr virus-associated gastric carcinoma.

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Southern blot hybridization法またはDot blot hybridization法によりHPV-DNAが検出されたが, ISH法ではHPV-DNAを検出できなかった27例の子宮頸部擦過細胞診材料 (Papanicolaou染色戻し標本) と同時期に採取された生検材料を用いin 法を行い以下の結果を得た.
1) 細胞診材料においてin 法で27例中16例 (59.3%) からHPV-DNAを検出した.
タイプ別HPVーDNAの検出率は, HPVpU-31Bでは, type6, 11群で5例中、3例 (60.0%), HPVpU-1Mでは, type16, 18群で14例中7例 (50.0%), type31, 33, 35群で8例中6例 (75.0%) であった.
2) HPV-DNAの分布は, 異型細胞を中心に一部正常細胞の核内に散在性に認められた.ただし, 正常細胞に陽性シグナルを得た症例はCIN1 (1例), CIN2 (1例) の2例であった.
3) 生検材料では27例中20例 (74.1%) でHPV-DNAを検出した.また細胞診材料との一致例は27例中24例 (88.9%), 不一致例は27例中3例 (11.1%) であった.
in 法は, 細胞診材料においても応用が可能で, ISH法に比べ感度が優れていることは, 今後の細胞形態学的遺伝子解析に大きく貢献するものと考える.

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In situ hybridization has been used for the detection of viral and bacterial mRNA and DNA which has infected tissue sections, but the sensitivity of this method is very limited. In this paper, a new, sensitive and simple non-isotopic in situ hybridization method in combination with a polymerise chain reaction (PCR) technique is described, in which the genomic or viral DNA can be amplified directly on formalin-fixed and paraffin-embedded tissue sections. As low as two copies of target DNA per cell can be detected by this in situ hybridization after in . The essential modification concerns a two step cycling PCR procedure that was applied using single primer pairs. The first step, annealing, was achieved at a low temperature for a relatively long time. The second step was performed at a high temperature for a short period of time. This procedure greatly decreased the non-specific amplification of DNA and increased the sensitivity of detection.

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口腔扁平上皮癌におけるヒトパピローマウイルス(HPV)の局在を明らかにするために, in 法を適用して高感度な検出法を開発した.ホルマリン固定パラフィン包埋切片を使用し, HPVのL1遺伝子をターゲットにしてMY 09-MY 11プライマーを用いてin 分析に影響する各種因子, すなわちthermal cyclerの種類(液体用と組織用), スライドガラスの処理, 組織の固定時間, 切片の消化酵素(pepsin, trypsin, protease K)処理と時間, PCR反応液の調製, 切片の乾燥防御, PCR増幅酵素の種類, PCRの回数, stringent washの時間, hot startの差異など諸条件を検討し, さらにin situ hybridization(ISH)との感度比較を行った.なおPCR時にはdigoxigenin(DIG)標識ヌクレオチド(DIG-11-dUTP)を使用, 染色時にはalkaline phosphatase標識抗digoxigenin抗体Fab fragmentsおよび基質と発色剤に5-bromo-4-chloro-3-indolyl-phosphate(BCIP)と4-nitroblue tetrazolium chloride(NBT)を使用し高感度染色を行った.その結果, 至適条件は, 有機シラン処理のスライドガラスに貼付した6枚の切片を使用して6種のpepsin消化時間で反応させ, 水洗後風乾してPCR反応液を載せシールしたのち, 90℃7分熱変性し, 30回のサイクル数で94℃1分の熱変性, 55℃2分のアニーリング, 72℃2分の伸張を行い, DNAを増幅したのち, さらに切片を50℃で30分間stringent washして免疫組織化学的染色を行うことであった.この条件下で扁平上皮癌12例の全例においてHPV DNAが検出できた.In は, ISHよりも検出頻度ならびに反応の強度いずれもが高感度を示し, 陽性反応はkoilocytosisを示す細胞, 形態学的に正常に見える細胞および腫瘍細胞にみられ, その局在は核内に全体的にあるいは散在的に存在するもの, エピソームとして核内外に顆粒状に存在するもの, および核と隣接する核外に存在するものとして観察された.以上のことからin 分析法の確立は今後HPVの発癌性の研究について有用な手段となることが示唆される.

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The abundance of denitrifying bacteria in soil has been determined primarily by the conventional most probable number (MPN) method. We have developed a single-cell identification technique that is culture-independent, direct in , to enumerate denitrifying bacteria in soils. The specificity of this method was evaluated with six species of denitrifying bacteria using nirK as the target gene; Escherichia coli was used as a negative control. Almost all (97.3%-100%) of the nirK-type denitrifying bacteria (Agromonas oligotrophica, Alcaligenes faecalis, Achromobacter denitrificans, Bradyrhizobium japonicum, and Pseudomonas chlororaphis) were detected by direct in , whereas no E. coli cells and only a few cells (2.4%) of nirS-type denitrifying bacteria (Pseudomonas aeruginosa) were detected. Numbers of denitrifying bacteria in upland and paddy soil samples quantified by this method were 3.3 × 10⁸ to 2.6 × 10⁹ cells g^-1 dry soil. These values are approximately 1,000 to 300,000 times higher than those estimated by the MPN method. These results suggest that direct in is a better tool for quantifying denitrifying bacteria in soil than the conventional MPN method.

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We developed a highly sensitive detection system for human papillomavirus (HPV) in order to elucidate the localization of HPV in formalin-fixed, Paraffin-embedded sections from squamous cell carcinoma (SCC) using in situ polymerase chain reaction (PCR) primers (MY 09 and MY 11). Various parameters such as thermal cycler apparatus, pepsin treatment, PCR reagent, stringency, PCR reaction condition, and immunohistochemistry were examined. Under optimal conditions, in analysis revealed the specific presence of HPV DNA in all cases of SCC and their localizations were observed in the nuclei and cytoplasms of koilocytes, SCC cells and epithelial cells as forms of integrated HPV DNA and episomes. These results suggest that the establishment of a highly accurate detection system for HPV will be very useful in carcinogenesis research.

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組織あるいは細胞内の標的とする遺伝子の局在を可視化するin について,その前処理として行う組織固定法,蛋白消化処理法, DNA損傷部位のマスキング法などの至適反応条件を検討した。BALB/C mouseの肝組織についてKi-ras癌遺伝子のエクソン部分を標的とするプライマーを用い,逆転写反応(RT)とDNAポリメラーゼ連鎖反応(PCR)の両方を触媒するrTth DNAポリメラーゼを使用して検討した結果,等張緩衝ホルマリン液による浸漬固定は15〜24時間が, pepsin処理は2mg/ml 0.1N HClの濃度で15〜17分間が最適条件である事が分かった。更にPCRの前に,ホルマリン及びパラフィン包埋時に生じたと考えられるDNA鎖損傷部位からのDNA合成反応を抑制するため, ddTTPを用いたDNA合成開始部位のマスキングを行う必要があることを明らかにした。

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