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1. 发明授权

US09347944B2 Method for detecting pneumonia causative bacteria using nucleic acid chromatography 有权
标题翻译：使用核酸色谱法检测肺炎致病菌的方法
公开(公告)号：US09347944B2
公开(公告)日：2016-05-24
申请号：US13637815
申请日：2011-03-30
申请人： Mutsunori Shirai , Takayuki Ezaki , Tsukasa Hayashi , Takeshi Ujiiye , Makoto Ganaha , Shigekazu Yamamoto
发明人： Mutsunori Shirai , Takayuki Ezaki , Tsukasa Hayashi , Takeshi Ujiiye , Makoto Ganaha , Shigekazu Yamamoto
IPC分类号： C12Q1/68 , G01N33/569
CPC分类号： G01N33/56933 , C12Q1/6816 , C12Q1/6865 , C12Q1/689 , C12Q2600/112 , C12Q2600/156 , G01N33/56911 , G01N33/56916 , G01N33/56938 , G01N33/56944 , Y02A50/451 , C12Q2565/137 , C12Q2525/143 , C12Q2537/125 , C12Q2537/143 , C12Q2563/149 , C12Q2565/625
摘要： Provided are a method and a kit for accurately and rapidly detecting ten types of targeting pneumonia bacteria: Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus. A set of primer pairs directed to their respective target regions contained in the DnaJ gene, etc., of the ten types of pneumonia causative bacteria is designed for the ten bacterial strains and used to amplify gene products. A set of bacterial strain-specific probe pairs is further designed for the ten bacterial strains such that the probe pairs hybridize with the amplification products via sequences in the respective target regions differing from the sequences hybridized by the set of primer pairs. A first probe-bound labeled high molecular carrier in which plural types of first probes for the pneumonia bacteria are bound to a labeled high molecular carrier and a solid-phase second probe-carrying developing support are used as the set of probe pairs to perform nucleic acid chromatography.
摘要翻译：提供了用于准确和快速检测十种靶向性肺炎细菌的方法和试剂盒：肺炎链球菌，流感嗜血杆菌，肺炎支原体，肺炎衣原体，嗜肺军团菌，肺炎克雷伯菌，铜绿假单胞菌，卡他莫拉菌，耐甲氧西林金黄色葡萄球菌（MRSA））和金黄色葡萄球菌（Staphylococcus aureus）。针对十种类型肺炎致病细菌的DnaJ基因等中包含的各自靶区域的一组引物对设计用于十种细菌菌株并用于扩增基因产物。针对10个细菌菌株进一步设计了一组细菌菌株特异性探针对，使得探针对通过与由该组引物对杂交的序列不同的各个靶区域中的序列与扩增产物进行杂交。使用第一探针结合标记的高分子载体，其中用于肺炎细菌的多种类型的第一探针与标记的高分子载体结合，并且使用固相第二探针携带的显影载体作为探针对的一组以进行核酸酸性色谱。

2. 发明申请

US20130023443A1 METHOD FOR DETECTING PNEUMONIA CAUSATIVE BACTERIA USING NUCLEIC ACID CHROMATOGRAPHY 有权
标题翻译：使用核酸色谱检测肺炎球菌引起的细菌的方法
公开(公告)号：US20130023443A1
公开(公告)日：2013-01-24
申请号：US13637815
申请日：2011-03-30
申请人： Mutsunori Shirai , Takayuki Ezaki , Tsukasa Hayashi , Takeshi Ujiiye , Makoto Ganaha , Shigekazu Yamamoto
发明人： Mutsunori Shirai , Takayuki Ezaki , Tsukasa Hayashi , Takeshi Ujiiye , Makoto Ganaha , Shigekazu Yamamoto
IPC分类号： C40B30/04 , C40B40/06
CPC分类号： G01N33/56933 , C12Q1/6816 , C12Q1/6865 , C12Q1/689 , C12Q2600/112 , C12Q2600/156 , G01N33/56911 , G01N33/56916 , G01N33/56938 , G01N33/56944 , Y02A50/451 , C12Q2565/137 , C12Q2525/143 , C12Q2537/125 , C12Q2537/143 , C12Q2563/149 , C12Q2565/625
摘要： Provided are a method and a kit for accurately and rapidly detecting ten types of targeting pneumonia bacteria: Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus. A set of primer pairs directed to their respective target regions contained in the DnaJ gene, etc., of the ten types of pneumonia causative bacteria is designed for the ten bacterial strains and used to amplify gene products. A set of bacterial strain-specific probe pairs is further designed for the ten bacterial strains such that the probe pairs hybridize with the amplification products via sequences in the respective target regions differing from the sequences hybridized by the set of primer pairs. A first probe-bound labeled high molecular carrier in which plural types of first probes for the pneumonia bacteria are bound to a labeled high molecular carrier and a solid-phase second probe-carrying developing support are used as the set of probe pairs to perform nucleic acid chromatography.
摘要翻译：提供了用于准确和快速检测十种靶向性肺炎细菌的方法和试剂盒：肺炎链球菌，流感嗜血杆菌，肺炎支原体，肺炎衣原体，嗜肺军团菌，肺炎克雷伯菌，铜绿假单胞菌，卡他莫拉菌，耐甲氧西林金黄色葡萄球菌（MRSA））和金黄色葡萄球菌（Staphylococcus aureus）。针对十种类型肺炎致病细菌的DnaJ基因等中包含的各自靶区域的一组引物对设计用于十种细菌菌株并用于扩增基因产物。针对10个细菌菌株进一步设计了一组细菌菌株特异性探针对，使得探针对通过与由该组引物对杂交的序列不同的各个靶区域中的序列与扩增产物进行杂交。使用第一探针结合标记的高分子载体，其中用于肺炎细菌的多种类型的第一探针与标记的高分子载体结合，并且使用固相第二探针携带的显影载体作为探针对的一组以进行核酸酸性色谱。

3. 发明授权

US08435742B2 Method and kit for detection/quantification of target RNA 有权
标题翻译：靶RNA的检测/定量的方法和试剂盒
公开(公告)号：US08435742B2
公开(公告)日：2013-05-07
申请号：US12739978
申请日：2008-11-04
申请人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
发明人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
IPC分类号： C12Q1/68 , C12P19/34 , C12N9/00 , C07H21/04
CPC分类号： C12Q1/6809 , C12Q2565/537 , C12Q2525/143 , C12Q2521/107
摘要： [Object] It is to provide a method and a kit capable of detecting or quantifying a target RNA simply and rapidly from trace amounts of RNA in a sample, in a case such as when one or more kinds of pathogenic microorganisms are to be detected or quantified.[Solving Means] The method comprises the steps of 1) synthesizing cDNA from a sample containing the target RNA using a liquid-phase primer having a promoter sequence and a reverse transcriptase to obtain a cDNA-RNA complex, 2) degrading the RNA of the complex, 3) synthesizing a double-stranded DNA via the cDNA obtained in the step 2) and the solid-phase primer, 4) synthesizing RNA from the double-stranded DNA, 5) synthesizing cDNA via the RNA obtained in the step 4) and the solid-phase primer to obtain a cDNA-RNA complex, 6) degrading the RNA of the complex obtained in step 5), 7) synthesizing a double-stranded DNA via the cDNA obtained in the step 6) and the liquid-phase primer, and 8) quantifying the double-stranded DNAs obtained in the steps 3) and 7). These steps can be performed in a single reaction solution.
摘要翻译：本发明提供一种方法和试剂盒，其能够在样品中的痕量RNA中简单快速地检测或定量靶RNA，例如当检测到一种或多种病原微生物或量化 [解决方法]该方法包括以下步骤：1）使用具有启动子序列和逆转录酶的液相引物合成来自含有靶RNA的样品的cDNA，得到cDNA-RNA复合物，2）降解复合物，3）通过步骤2）和固相引物获得的cDNA合成双链DNA，4）从双链DNA合成RNA，5）通过步骤4）得到的RNA合成cDNA，和固相引物以获得cDNA-RNA复合物，6）降解步骤5）中获得的复合物的RNA，7）通过步骤6）中得到的cDNA合成双链DNA和液相引物，和8）定量在步骤3）和7）中获得的双链DNA。这些步骤可以在单个反应溶液中进行。

4. 发明授权

US07094405B1 Peptides which elicit a high neutralizing antibody titer, cytotoxic T lymphocyte response and T helper cell response in a broad range of MHC type recipients 失效
标题翻译：在广泛的MHC型受体中引发高中和抗体滴度，细胞毒性T淋巴细胞反应和T辅助细胞应答的肽
公开(公告)号：US07094405B1
公开(公告)日：2006-08-22
申请号：US09455076
申请日：1999-12-06
申请人： Jay A. Berzofsky , Jeffrey D. Ahlers , C. David Pendleton , Peter Nara , Mutsunori Shirai
发明人： Jay A. Berzofsky , Jeffrey D. Ahlers , C. David Pendleton , Peter Nara , Mutsunori Shirai
IPC分类号： A61K39/21
CPC分类号： C07K14/005 , A61K38/03 , A61K38/55 , A61K38/556 , A61K39/00 , A61K39/12 , A61K39/21 , A61K2039/545 , A61K2039/55516 , A61K2039/55566 , A61K2039/55577 , A61K2039/57 , A61K2039/645 , C07K2319/00 , C12N2740/16122 , C12N2740/16134 , Y02A50/412 , A61K2300/00
摘要： Peptide constructs comprised of multideterminant T helper peptides from the envelope glycoprotein of HIV previously identified to induce proliferative responses in four different haplotypes of mice and IL-2 responses in 52-73% of HIV positive, flu positive patients (cluster peptides), were co-linearly synthesized with the peptide 18 of the V3 loop of HIV-1 gp 160, corresponding to the principal neutralizing determinant of HIV-IIIB and also shown to contain a dominant CTL epitope. Cognate help for peptide 18 antibody was elicited following a single immunization in all strains of mice which had previously responded to a T cell epitope encompassed by the peptides. In two strains of mice, the level of neutralizing antibody achieved was comparable to levels adequate for protection from homologous viral challenge in chimpanzees. After a single boost, much higher antibody titers for 90% neutralization in the range of 1:1000 to 1:16,000 were achieved. Spleen cells from mice of three distinct MHC haplotypes sharing the Dd class I MHC molecule but with different class II molecules, immunized with the compound peptides, exhibited enhanced gp160-specific CTL activity.
摘要翻译：由先前鉴定为在四种不同单倍型小鼠中诱导增殖反应的HIV的多重决定子T辅助肽和52-73％的HIV阳性患者（簇肽）中的IL-2应答组成的多肽构建体由co 与HIV-1 gp 160的V3环的肽18线性合成，对应于HIV-IIIB的主要中和决定簇，并且还显示含有显性CTL表位。在先前对肽包含的T细胞表位作出反应的所有小鼠菌株中进行单次免疫后，引发了针对肽18抗体的协同帮助。在两种小鼠中，达到的中和抗体水平与足以保护黑猩猩同源病毒攻击的水平相当。在单次加强后，实现了在1:1000至1：16,000范围内高达90％中和的高得多的抗体滴度。来自共享第一类MHC分子但具有用化合物肽免疫的不同II类分子的三种不同MHC单元型的小鼠的脾细胞表现出增强的gp160特异性CTL活性。

5. 发明授权

US06294322B1 Multideterminant peptides that elicit helper T-lymphocyte cytotoxic T-lymphocyte and neutralizing antibody responses against HIV-1 失效
标题翻译：引起辅助T淋巴细胞细胞毒性T淋巴细胞和中和抗HIV-1抗体的多决定肽
公开(公告)号：US06294322B1
公开(公告)日：2001-09-25
申请号：US08060988
申请日：1993-05-14
申请人： Jay A. Berzofsky , Jeffrey D. Ahlers , C. David Pendleton , Peter Nara , Mutsunori Shirai
发明人： Jay A. Berzofsky , Jeffrey D. Ahlers , C. David Pendleton , Peter Nara , Mutsunori Shirai
IPC分类号： C12Q170
CPC分类号： C07K14/005 , A61K38/03 , A61K38/55 , A61K38/556 , A61K39/00 , A61K39/12 , A61K39/21 , A61K2039/545 , A61K2039/55516 , A61K2039/55566 , A61K2039/55577 , A61K2039/57 , A61K2039/645 , C07K2319/00 , C12N2740/16122 , C12N2740/16134 , Y02A50/412 , A61K2300/00
摘要： Peptide constructs comprised of multideterminant T helper peptides from the envelope glycoprotein of HIV previously identified to induce proliferative responses in four different haplotypes of mice and IL-2 responses in 52-73% of HIV positive, flu positive patients (cluster peptides), were co-linearly synthesized with the peptide 18 of the V3 loop of HIV-1 gp 160, corresponding to the principal neutralizing determinant of HIV-IIIB and also shown to contain a dominant CTL epitope. Cognate help for peptide 18 antibody was elicited following a single immunization in all strains of mice which had previously responded to a T cell epitope encompassed by the peptides. In two strains of mice, the level of neutralizing antibody achieved was comparable to levels adequate for protection from homologous viral challenge in chimpanzees. After a single boost, much higher antibody titers for 90% neutralization in the range of 1:1000 to 1:16,000 were achieved. Spleen cells from mice of three distinct MHC haplotypes sharing the Dd class I MHC molecule but with different class II molecules, immunized with the compound peptides, exhibited enhanced gp160-specific CTL activity.
摘要翻译：由先前鉴定为在四种不同单倍型小鼠中诱导增殖反应的HIV的多重决定子T辅助肽和52-73％的HIV阳性患者（簇肽）中的IL-2应答组成的多肽构建体由co 与HIV-1 gp 160的V3环的肽18线性合成，对应于HIV-IIIB的主要中和决定簇，并且还显示含有显性CTL表位。在先前对肽包含的T细胞表位作出反应的所有小鼠菌株中进行单次免疫后，引发了针对肽18抗体的协同帮助。在两种小鼠中，达到的中和抗体水平与足以保护黑猩猩同源病毒攻击的水平相当。在单次加强后，实现了在1:1000至1：16,000范围内高达90％中和的高得多的抗体滴度。来自共享Dd I类MHC分子但具有用化合物肽免疫的不同II类分子的三种不同MHC单元型的小鼠的脾细胞表现出增强的gp160特异性CTL活性。

6. 发明授权

US08440195B2 Inhibition of CD69 for treatment of inflammatory conditions 有权
标题翻译：抑制CD69治疗炎症状况
公开(公告)号：US08440195B2
公开(公告)日：2013-05-14
申请号：US12945153
申请日：2010-11-12
申请人： Toshinori Nakayama , Akihiro Hasegawa , Mutsunori Shirai
发明人： Toshinori Nakayama , Akihiro Hasegawa , Mutsunori Shirai
IPC分类号： A61K39/395
CPC分类号： A61K39/3955 , A61K31/00 , A61K45/06 , A61K47/6849 , A61K2039/505 , C07K16/2851 , C07K16/2896
摘要： A method of treating or reducing at least one inflammatory condition or the susceptibility to at least one inflammatory condition is provided involving administering at least one CD69 antagonist to a subject, wherein the subject has been diagnosed with at least one inflammatory condition, or a susceptibility to the same. CD69 antagonists can include one or more of an anti-CD69 antibody, an anti-CD69 aptamer, a CD69 mRNA antagonist, and a small molecule pharmaceutical.
摘要翻译：提供治疗或减少至少一种炎性病症或对至少一种炎性病症的易感性的方法，包括对受试者施用至少一种CD69拮抗剂，其中所述受试者已被诊断患有至少一种炎性病症或易感性一样。 CD69拮抗剂可以包括一种或多种抗CD69抗体，抗CD69适体，CD69mRNA拮抗剂和小分子药物。

7. 发明申请

US20120121503A1 Inhibition Of CD69 For Treatment Of Inflammatory Conditions 有权
标题翻译：抑制CD69用于治疗炎症状况
公开(公告)号：US20120121503A1
公开(公告)日：2012-05-17
申请号：US12945153
申请日：2010-11-12
申请人： Toshinori Nakayama , Akihiro Hasegawa , Mutsunori Shirai
发明人： Toshinori Nakayama , Akihiro Hasegawa , Mutsunori Shirai
IPC分类号： A61K39/395 , A61P1/16 , A61K31/195 , A61P29/00 , A61K51/10 , A61K31/713
CPC分类号： A61K39/3955 , A61K31/00 , A61K45/06 , A61K47/6849 , A61K2039/505 , C07K16/2851 , C07K16/2896
摘要： A method of treating or reducing at least one inflammatory condition or the susceptibility to at least one inflammatory condition is provided involving administering at least one CD69 antagonist to a subject, wherein the subject has been diagnosed with at least one inflammatory condition, or a susceptibility to the same. CD69 antagonists can include one or more of an anti-CD69 antibody, an anti-CD69 aptamer, a CD69 mRNA antagonist, and a small molecule pharmaceutical.
摘要翻译：提供治疗或减少至少一种炎性病症或对至少一种炎性病症的易感性的方法，包括对受试者施用至少一种CD69拮抗剂，其中所述受试者已被诊断患有至少一种炎性病症或易感性一样。 CD69拮抗剂可以包括一种或多种抗CD69抗体，抗CD69适体，CD69mRNA拮抗剂和小分子药物。

8. 发明授权

US5980899A Identification of peptides that stimulate hepatitis C virus specific cytotoxic T cells 失效
标题翻译：鉴定刺激丙型肝炎病毒特异性细胞毒性T细胞的肽
公开(公告)号：US5980899A
公开(公告)日：1999-11-09
申请号：US894063
申请日：1992-06-10
申请人： Jay A. Berzofsky , Mutsunori Shirai , Toshitaka Akatsuka , Stephen M. Feinstone
发明人： Jay A. Berzofsky , Mutsunori Shirai , Toshitaka Akatsuka , Stephen M. Feinstone
IPC分类号： G01N33/53 , A61K38/00 , A61K39/00 , A61K39/29 , A61P31/12 , A61P37/00 , C07K7/06 , C07K7/08 , C07K14/18 , G01N33/569 , G01N33/576 , A61K39/12 , C07K2/00
CPC分类号： C07K14/005 , A61K39/00 , C12N2770/24222
摘要： The cytotoxic T cell response to the protein encoded by the NS5 region of hepatitis C virus was determined using 28 peptides from NS5 which were selected by an amphipathicity algorithm as candidates for T cell epitopes. In BALB/c mice, a single relatively conserved epitope represented by a 16-residue synthetic peptide was presented by D.sup.d class I major histocompatibility complex (MHC) molecules to conventional CD4.sup.- CD8.sup.+ CTL. An exemplary peptide, which represents amino acid residues 2422-2437 of the polyprotein of the Chiron HCV1 isolate, had the amino acid sequence MSYSWTGALVTPCAAE [SEQ ID NO: 1]. A CTL line specific for this peptide recognized the two known natural variants of this NS5 sequence, each with conservative substitutions. Thus, CTL can recognize the product of the HCV NS5 gene, the probable RNA polymerase, in association with class I MHC molecules on model target cells and may recognize the same epitope on hepatocytes or any other cells infected with the virus.
摘要翻译：使用来自NS5的28个肽来确定由丙型肝炎病毒的NS5区编码的蛋白质的细胞毒性T细胞应答，其通过两亲性算法选择作为T细胞表位的候选物。在BALB / c小鼠中，通过Dd I类主要组织相容性复合物（MHC）分子向常规CD4-CD8 + CTL提供了由16个残基合成肽表示的单个相对保守的表位。表示Chiron HCV1分离物的多蛋白的氨基酸残基2422-2437的示例性肽具有氨基酸序列MSYSWTGALVTPCAAE [SEQ ID NO：1]。该肽特异的CTL系识别了NS5序列的两个已知天然变体，每个具有保守取代。因此，CTL可以识别HCV NS5基因（可能的RNA聚合酶）与模型靶细胞上的I类MHC分子相关联的产物，并且可以识别肝细胞或感染病毒的任何其它细胞上的相同表位。

9. 发明授权

US5976541A Potent peptide for stimulation of cytotoxic T lymphocytes specific for the HIV-1 envelope 失效
标题翻译：用于刺激特异于HIV-1信封的细胞毒性T淋巴细胞的强力肽
公开(公告)号：US5976541A
公开(公告)日：1999-11-02
申请号：US847311
申请日：1992-03-06
申请人： Jay A. Berzofsky , Toshiyuki Taskeshita , Mutsunori Shirai , C. David Pendleton , Steven Kozlowski , David H. Margulies
发明人： Jay A. Berzofsky , Toshiyuki Taskeshita , Mutsunori Shirai , C. David Pendleton , Steven Kozlowski , David H. Margulies
IPC分类号： A61K38/03 , A61K38/55 , A61K39/00 , A61P31/18 , C07K7/06 , C07K7/08 , C07K14/16 , C12N15/48 , A61K39/21 , A61K39/12 , A61K39/38 , C12N15/00
CPC分类号： C07K14/005 , A61K38/03 , A61K38/55 , A61K38/556 , A61K39/00 , C12N2740/16122
摘要： Peptides having high activity in the eliciting of a cytotoxic T lymphocyte response to the HIV-1 envelope glycoprotein gpl60 are described. The activation of 12-15 residue peptides by proteolytic degradation to shorter peptides is shown as are general techniques for characterizing such activation processes.
摘要翻译：描述了在引起对HIV-1包膜糖蛋白gp1660的细胞毒性T淋巴细胞反应中具有高活性的肽。通过蛋白水解降解到较短肽的12-15个残基肽的活化显示为表征这种活化过程的一般技术。

10. 发明申请

US20110053150A1 Method and Kit for Detection/Quantification of Target RNA 有权
标题翻译：靶RNA的检测/定量的方法和试剂盒
公开(公告)号：US20110053150A1
公开(公告)日：2011-03-03
申请号：US12739978
申请日：2008-11-04
申请人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
发明人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6809 , C12Q2565/537 , C12Q2525/143 , C12Q2521/107
摘要： [Object] It is to provide a method and a kit capable of detecting or quantifying a target RNA simply and rapidly from trace amounts of RNA in a sample, in a case such as when one or more kinds of pathogenic microorganisms are to be detected or quantified.[Solving Means] The method comprises the steps of 1) synthesizing cDNA from a sample containing the target RNA using a liquid-phase primer having a promoter sequence and a reverse transcriptase to obtain a cDNA-RNA complex, 2) degrading the RNA of the complex, 3) synthesizing a double-stranded DNA via the cDNA obtained in the step 2) and the solid-phase primer, 4) synthesizing RNA from the double-stranded DNA, 5) synthesizing cDNA via the RNA obtained in the step 4) and the solid-phase primer to obtain a cDNA-RNA complex, 6) degrading the RNA of the complex obtained in step 5), 7) synthesizing a double-stranded DNA via the cDNA obtained in the step 6) and the liquid-phase primer, and 8) quantifying the double-stranded DNAs obtained in the steps 3) and 7). These steps can be performed in a single reaction solution.
摘要翻译：本发明提供一种方法和试剂盒，其能够在样品中的痕量RNA中简单快速地检测或定量靶RNA，例如当检测到一种或多种病原微生物或量化 [解决方法]该方法包括以下步骤：1）使用具有启动子序列和逆转录酶的液相引物合成来自含有靶RNA的样品的cDNA，得到cDNA-RNA复合物，2）降解复合物，3）通过步骤2）和固相引物获得的cDNA合成双链DNA，4）从双链DNA合成RNA，5）通过步骤4）得到的RNA合成cDNA，和固相引物以获得cDNA-RNA复合物，6）降解步骤5）中获得的复合物的RNA，7）通过步骤6）中得到的cDNA合成双链DNA和液相引物，和8）定量在步骤3）和7）中获得的双链DNA。这些步骤可以在单个反应溶液中进行。

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