定量聚合酶链反应

摘要： 目的验证以问题为基础(PBL)教学法在提高学生处理聚合酶链反应(PCR)污染能力方面的作用。方法以PCR假阳性为实例,采用PBL教学法引导学员合理设立空白对照,通过重复试验探究污染来源。结果学员在思维引导帮助下逐步发现PCR污染来源于引物污染,并对试验方法及理论有了清晰的认识。结论以实例讲解为基础的PBL教学法可有效提高学生认识和处理PCR污染的能力。

摘要： 目的 通过比较提取效率选择新型冠状病毒核酸提取试剂盒,并考察方法对血浆的核酸检测适用性.方法 采用3种不同厂家核酸提取试剂盒对血浆中新型冠状病毒核酸进行提取,使用1种核酸检测试剂盒进行扩增和核酸检测.结果 核衣壳蛋白(nucleocapcid,N)基因片段和开放阅读框1a/b(open reading frame 1a/b,ORF1ab)基因片段标准曲线的线性相关系数均≥0.999.核酸提取试剂盒A对ORF1ab基因的回收率为91％,对N基因的回收率为38％;试剂盒B对ORF1ab基因的回收率为104％,对N基因的回收率为44％;试剂盒C对ORF1ab基因的回收率为0％,对N基因的回收率为12％.采用试剂盒A、B均能达到100％正确率,试剂盒C仅能达到62.5％正确率,误检均为假阴性.结论 采用提取试剂盒A、B和新型冠状病毒2019-nCoV核酸检测试剂盒进行核酸检测,均能满足新型冠状病毒肺炎康复者恢复期血浆中病毒核酸的检测需求.

摘要： 目前发现爱泼斯坦巴尔病毒(Epstein-Barr virus,EBV)与多种淋巴组织增生性疾病的关系越来越密切,主要包括EBV相关淋巴瘤、EBV阳性淋巴组织增殖性疾病(EBV+LPD)以及传染性单核细胞增多症(infectious mononucleosis,IM)等,以往研究认为EBV+LPD及IM的外周血EBV DNA有高拷贝数,而EBV相关淋巴瘤的外周血EBV DNA拷贝数一般不高,而近几年有不少文献报道EBV相关淋巴瘤外周血EBV DNA也出现高拷贝数,同时一些研究已经证实EBV DNA载量的检测不仅可以用于EBV相关淋巴组织增生性疾病的诊断,还可用于评价患者对治疗的反应及预后.该文主要对各种不同类型淋巴组织增生性疾病患者外周血EBV DNA定量检测做一综述,并探讨在治疗反应、预后判断中的价值和意义.

摘要： 病毒检测在病毒感染的预防、诊断、治疗及病毒性疫苗的质量控制等方面发挥着重要的作用.实时荧光定量PCR通过荧光信号实时检测目的 基因扩增数量,是目前实验室最常用的的病毒检测技术,具有灵敏度高、检测快速、污染小等优点.此文对实时荧光定量PCR技术在病毒感染的快速诊断、病毒核酸定量检测和病毒型别的鉴定三个方面的应用进行阐述.

摘要： 目的 分析健康献血人群人乳头瘤病毒(HPV)DNA载量,评估其潜在的临床意义.方法 采集207名健康献血者的血液,提取基因组DNA,采用GP5+/GP6+引物进行定量聚合酶链反应(PCR),同时对扩增的HPV L1基因140～150 bp的特异性片段进行测序,并进行基因分型.结果 207名献血者中,有14名(6.8％)献血者HPV DNA阳性,其中7名为单一型别HPV感染,7名为混合感染.所有阳性样本病毒载量为12.4～39.3拷贝/mL.结论 小部分健康人血液中存在低载量的HPV DNA,血液可能是HPV传播的新途径,不排除健康献血者存在潜在的生殖道或其他部位的HPV感染.

摘要： 在侵袭性真菌病中,毛霉菌病的发病率逐年上升,对免疫功能低下患者的健康构成重大威胁。毛霉菌病的快速鉴别诊断,并尽早开始有效的抗真菌治疗对于患者预后至关重要。目前缺少可早期检测毛霉菌感染的标准试验,毛霉菌病的诊断以培养和显微镜检查为金标准,为了建立一种早期快速地检测试验,检验人员进行了大量基于实时荧光定量PCR(qPCR)的分子诊断研究。本文对近年来毛霉菌病定量聚合酶链反应检测方法的研究进展进行综述。

摘要： Duan及其同事通过16S核糖体RNA基因测序,发现酒精使用障碍和酒精性肝炎(AH)患者胃肠道微生物组与对照组之间存在差异。在这些差异中,AH患者的肠球菌数量明显更多,占粪便分离细菌总量的5.59%,而对照组仅占0.023%。定量聚合酶链反应(q PCR)结果显示AH患者粪便肠球菌含量约为对照组的2700倍。此外,30%AH患者粪便样本中检测出编码溶细胞毒素的基因(由粪肠球菌产生)且对真核细胞和革兰阳性细菌具有活性作用。但30例酒精使用障碍患者中仅有1例上述基因检测阳性,25例对照病例该基因检测均为阴性。

摘要： Objective To verify the detection method for residual host cell DNA in enterovirus 71 inactivated vaccine (Vero cell) (EV71 vaccine) by magnetic bead based extraction combined with quantitative PCR (qPCR). Methods Residual host cell DNA in samples was extracted utilizing the specific binding of magnetic bead to DNA in protein solution. qPCR was performed on extracted DNA and serial diluted standards simultaneously. Residual DNA in samples was quantitatively analyzed according to the linear relationship between cycle threshold values and concentrations of standards. Results The standard detection range was 0.03-3 000.00 pg/reaction. The correlation coefficient of standard curve was >0.980, with amplification efficiency at 90.0％-110.0％. The recovery rates of spiked samples were50％-150％, and the relative standard deviation was <30％. All parameters of results were within the required range. Conclusions The magnetic bead based extraction method can solve technical difficulties in sample pretreatment for residual DNA assay. qPCR is a simple, rapid and accurate method for quantitation of residual DNA in EV71 vaccine. This method is suitable for quality control of EV71 vaccine in production, and may provide indication for quality control of other same cell based viral vaccines.%目的 对磁珠法结合定量PCR(quantitative PCR,qPCR)检测肠道病毒71型灭活疫苗(Vero细胞)(EV71疫苗)中宿主DNA残留量进行适用性验证及应用.方法 利用微磁珠与蛋白溶液中DNA的特异性结合,来提取样品中残留的宿主细胞DNA.将已知浓度的细胞DNA对照系列稀释后,作为标准品DNA与提取的DNA同时进行qPCR扩增.根据标准品DNA的循环阈值与浓度之间的线性关系,对未知样品中残留DNA进行定量分析.结果 标准品检测范围在0.03～3 000.00 pg/反应.该方法的标准曲线决定系数≥0.980,扩增效率为90.0％～110.0％.质控样品回收率为50％～150％,相对标准偏差均小于30％.实验结果的各项参数均在要求范围内.结论 磁珠法可解决残留DNA检测中样品前处理的技术难点,qPCR能够简便、快速、准确地对EV71疫苗生产过程中的DNA残留量进行定量测定.该法适用于EV71疫苗中DNA残留量的检测及疫苗生产过程和其成品的质量控制,对其他采用同样细胞基质的病毒性疫苗质量控制具有借鉴意义.

摘要： 创伤性脑损伤（TBI）是致死和致残的主要原因。微小RNA（miRs）是在转录后水平负向调控基因表达的一种非编码小RNA-miRs可能是神经元凋亡的关键调节因子，但其对TBI后继发性损伤的作用仍不明确。有学者进行了一项动物研究，采用miR阵列和定量聚合酶链反应（qPCR）检测大脑皮质控制性损伤（cci）小鼠72h内miRs的变化。

摘要： 目的：探讨酶联免疫吸附试验法(ELISA)检测乙肝两对半的应用价值。方法将我院2013年12月至2015年1月就诊时做过乙肝两对半检测疑似有乙肝的128例患者作为研究对象，分别对患者采取酶联免疫吸附试验法和定量聚合酶链反应(Q-PCR)方法实施检测，比较两种方法检测的结果。结果采用定量聚合酶链反应检测方法检出率为94.53%(121/128)，采用取酶联免疫吸附试验法检出率为88.28(113/128)，两种检出率比较，无明显差异(P<0.05)。结论酶联免疫吸附试验法与定量聚合酶链反应检测方法在乙肝两对半的检测中检出率无明显差异，检出率均高，但是采用定量聚合酶链反应检测相对聚合酶链反应检测方方法来说，具有操作简单的优势，更适用于急诊中的检测。

摘要： Telomere is the end of a chromosome, which is a specialized structure involved in the replication and stability of the chromosome. Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of telomere length[1,2]. Cawthon[1] designed oligonucleotide primers tel1 and tel2 that hybridize to the TTAGGG and CCCTAA repeats and used AmpliTaq Gold DNA polymerase on the Applied Biosystems Prism 7700 sequence detection system. Gil and Coetzer[2] modified Cawthon's method and described the parameters and reagents required to measure telomere length using the Roche LightCycler. However, the advantage of the primers tel1b and tel 2b was not mentioned. In recent years, β-globin is used more often than 36B4 as the reference single-copy gene[e.g., 3-5], but the reason of giving up 36B4 was not put forward. Moreover, the real-time thermal cycler used in our laboratory, as well as in numerous laboratories worldwide, is the Takara PCR Thermal Cycler Dice Real Time System (TP800) using the modified Takara Ex Taq HS. In the beginning we attempted the assay by applying the conditions published for the Prism 7700 and LightCycler onto the TP800. However, owing to different reagents and inherent differences between these instruments, substantial optimization was required for the technique to be applied to the TP800. Here we describe the reagents and conditions required to measure telomere length using the TP800.

摘要： Telomere is the end of a chromosome, which is a specialized structure involved in the replication and stability of the chromosome. Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of telomere length[1,2]. Cawthon[1] designed oligonucleotide primers tel1 and tel2 that hybridize to the TTAGGG and CCCTAA repeats and used AmpliTaq Gold DNA polymerase on the Applied Biosystems Prism 7700 sequence detection system. Gil and Coetzer[2] modified Cawthon's method and described the parameters and reagents required to measure telomere length using the Roche LightCycler. However, the advantage of the primers tel1b and tel 2b was not mentioned. In recent years, β-globin is used more often than 36B4 as the reference single-copy gene[e.g., 3-5], but the reason of giving up 36B4 was not put forward. Moreover, the real-time thermal cycler used in our laboratory, as well as in numerous laboratories worldwide, is the Takara PCR Thermal Cycler Dice Real Time System (TP800) using the modified Takara Ex Taq HS. In the beginning we attempted the assay by applying the conditions published for the Prism 7700 and LightCycler onto the TP800. However, owing to different reagents and inherent differences between these instruments, substantial optimization was required for the technique to be applied to the TP800. Here we describe the reagents and conditions required to measure telomere length using the TP800.

摘要： Telomere is the end of a chromosome, which is a specialized structure involved in the replication and stability of the chromosome. Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of telomere length[1,2]. Cawthon[1] designed oligonucleotide primers tel1 and tel2 that hybridize to the TTAGGG and CCCTAA repeats and used AmpliTaq Gold DNA polymerase on the Applied Biosystems Prism 7700 sequence detection system. Gil and Coetzer[2] modified Cawthon's method and described the parameters and reagents required to measure telomere length using the Roche LightCycler. However, the advantage of the primers tel1b and tel 2b was not mentioned. In recent years, β-globin is used more often than 36B4 as the reference single-copy gene[e.g., 3-5], but the reason of giving up 36B4 was not put forward. Moreover, the real-time thermal cycler used in our laboratory, as well as in numerous laboratories worldwide, is the Takara PCR Thermal Cycler Dice Real Time System (TP800) using the modified Takara Ex Taq HS. In the beginning we attempted the assay by applying the conditions published for the Prism 7700 and LightCycler onto the TP800. However, owing to different reagents and inherent differences between these instruments, substantial optimization was required for the technique to be applied to the TP800. Here we describe the reagents and conditions required to measure telomere length using the TP800.

摘要： Telomere is the end of a chromosome, which is a specialized structure involved in the replication and stability of the chromosome. Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of telomere length[1,2]. Cawthon[1] designed oligonucleotide primers tel1 and tel2 that hybridize to the TTAGGG and CCCTAA repeats and used AmpliTaq Gold DNA polymerase on the Applied Biosystems Prism 7700 sequence detection system. Gil and Coetzer[2] modified Cawthon's method and described the parameters and reagents required to measure telomere length using the Roche LightCycler. However, the advantage of the primers tel1b and tel 2b was not mentioned. In recent years, β-globin is used more often than 36B4 as the reference single-copy gene[e.g., 3-5], but the reason of giving up 36B4 was not put forward. Moreover, the real-time thermal cycler used in our laboratory, as well as in numerous laboratories worldwide, is the Takara PCR Thermal Cycler Dice Real Time System (TP800) using the modified Takara Ex Taq HS. In the beginning we attempted the assay by applying the conditions published for the Prism 7700 and LightCycler onto the TP800. However, owing to different reagents and inherent differences between these instruments, substantial optimization was required for the technique to be applied to the TP800. Here we describe the reagents and conditions required to measure telomere length using the TP800.

摘要： Telomere is the end of a chromosome, which is a specialized structure involved in the replication and stability of the chromosome. Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of telomere length[1,2]. Cawthon[1] designed oligonucleotide primers tel1 and tel2 that hybridize to the TTAGGG and CCCTAA repeats and used AmpliTaq Gold DNA polymerase on the Applied Biosystems Prism 7700 sequence detection system. Gil and Coetzer[2] modified Cawthon's method and described the parameters and reagents required to measure telomere length using the Roche LightCycler. However, the advantage of the primers tel1b and tel 2b was not mentioned. In recent years, β-globin is used more often than 36B4 as the reference single-copy gene[e.g., 3-5], but the reason of giving up 36B4 was not put forward. Moreover, the real-time thermal cycler used in our laboratory, as well as in numerous laboratories worldwide, is the Takara PCR Thermal Cycler Dice Real Time System (TP800) using the modified Takara Ex Taq HS. In the beginning we attempted the assay by applying the conditions published for the Prism 7700 and LightCycler onto the TP800. However, owing to different reagents and inherent differences between these instruments, substantial optimization was required for the technique to be applied to the TP800. Here we describe the reagents and conditions required to measure telomere length using the TP800.

摘要： 本发明涉及在PCR产物的末端上互补结合探针的情况下，利用抑制DNA聚合酶的5'‑瓣状内切核酸酶(FEN)活性的特性来检测单核苷酸多态性(SNP)的方法。更具体地涉及将实时聚合酶链反应(real‑time PCR)中使用的探针与PCR产物末端进行互补结合的情况下，确认抑制针对探针的耐热性DNA聚合酶的5'‑瓣状内切核酸酶活性的特性，并利用其特性来使待检测的单核苷酸多态性(SNP)部位位于探针的5'‑末端部位时，根据等位基因(allele)来诱导5'‑瓣状的形成，从而可有效地检测单核苷酸多态性(SNP)的方法。

摘要： 本发明为一种一步法实时荧光定量RT‑PCR检测人细胞角蛋白7 试剂盒，该试剂盒以mRNA为模板，使反转录和实时荧光定量PCR在同一反应体系中进行，反应过程中无需打开管盖，避免了交叉污染，并可以精确定量检测标本中人细胞角蛋白7(CK7) 的mRNA表达量。该试剂盒可以用于临床及科研中检测患者外周血、淋巴结等标本中CK7的表达，用以辅助诊断、指导治疗结直肠癌及提示预后。

摘要： 本发明为一种实时荧光定量RT‑PCR检测人基质金属蛋白酶1的试剂盒，该试剂盒以mRNA为模板，使反转录和实时荧光定量PCR在同一反应体系中进行，反应过程中无需打开管盖，避免了交叉污染，并可以精确定量检测标本中人基质金属蛋白酶1 (MMP1) 的mRNA表达量。该试剂盒可以用于临床及科研中检测患者结直肠癌手术切除标本中MMP1的表达，用以结直肠癌患者辅助诊断、指导治疗是否存在肝转移的指标。

摘要： 本发明为一种实时荧光定量RT‑PCR检测人基质金属蛋白酶11的试剂盒，该试剂盒以mRNA为模板，使反转录和实时荧光定量PCR在同一反应体系中进行，反应过程中无需打开管盖，避免了交叉污染，并可以精确定量检测标本中人基质金属蛋白酶11(MMP11)的mRNA表达量。该试剂盒可以用于临床及科研中检测患者结直肠癌手术切除标本中MMP11的表达，用以结直肠癌患者的辅助诊断、指导治疗及提示预后。

摘要： 本发明为一种实时荧光定量RT‑PCR检测人miR‑1290试剂盒，该试剂盒以总mRNA为模板，使反转录和实时荧光定量PCR在同一反应体系中进行，反应过程中无需打开管盖，避免了交叉污染，并可以精确定量检测标本中人miR‑1290的表达量。该试剂盒可以用于临床及科研中检测患者外周血标本中miR‑1290的表达，用以结直肠癌辅助诊断、指导治疗、复发及提示预后。

摘要： 本发明为一种实时荧光定量RT‑PCR检测人上皮细胞粘附分子试剂盒，该试剂盒以mRNA为模板，使反转录和实时荧光定量PCR在同一反应体系中进行，反应过程中无需打开管盖，避免了交叉污染，并可以精确定量检测标本中人上皮细胞粘附分子(Ep‑CAM) 的mRNA表达量。该试剂盒可以用于临床及科研中检测患者外周血标本中EP‑CAM的表达，用以结直肠癌辅助诊断、指导治疗及提示预后。

摘要： 本发明涉及一种能够定量检测人、大鼠、小鼠等多个物种的ACTB基因表达的荧光定量体外扩增检测试剂盒；其中包含ACTB特异的引物、探针和耐热DNA聚合酶、dNTPs、模板DNA、镁离子、PCR缓冲液、超纯水等组成的PCR扩增缓冲反应体系，以及标准ACTB模板；其中所用引物和探针序列分别为：上游引物：SEQ ID NO 1；下游引物：SEQ ID NO 2；探针序列：SEQ ID NO 3；引物和探针浓度为每条0.25umol/L、Taq DNA聚合酶1.5U/50ul、dNTPs底物0.3mmol/L、模板cDNA若干、Mg++2.5mmol/L、缓冲液1XPCR；标准ACTB模板序列为：SEQ ID NO 4。

摘要： 本发明属生物技术领域，涉及定量RT-PCR检测试剂盒，具体是一种实时荧光定量逆转录聚合酶链反应检测的鸡高迁移率族蛋白B1的试剂盒。该试剂盒含有逆转录反应液、莫洛尼氏鼠白血病病毒逆转录酶、RNA酶抑制剂、聚合酶链反应液、耐热DNA聚合酶、标准品、对照品。本发明试剂盒通过逆转录(RT)mRNA样品得到cDNA，再结合实时荧光定量PCR检测技术，可以精确定量检测标本中鸡高迁移率族蛋白B1的mRNA表达量。该试剂盒可以用于鸡体内各组织脏器中HMGB1的表达分析，也可以用于检测不同状态下细胞中鸡HMGB1表达水平的变化。该发明为研究鸡HMGB1表达水平提供了一种快速、准确、可重复的检测方法。

摘要： 本发明涉及一种亚历山大藻荧光定量聚合酶链反应标准品的构建方法，通过提取A.catenella ACDH01的DNA，聚合酶链反应扩增A.catenella的18SrDNA到28S rDNA基因片段，并将扩增产物和T载体连接，构建重组质粒。经确认的重组质粒梯度稀释成聚合酶链式反应标准品；以5.8S-b5’和5.8S-b3’为引物，以重组质粒标准品为模板，real-time聚合酶链反应，根据反应中的临界循环数以及重组质粒标准品的梯度浓度对数值制备标准曲线。本发明建立了带有A.catenella的18S rDNA-28S rDNA基因片段的重组质粒real-time聚合酶链反应标准品，采用一对通用引物，可以准确、高效、快速地对待测的亚历山大藻real-time聚合酶链反应检测。

摘要： 本实用新型公开了一种实时荧光定量聚合酶链反应仪，涉及荧光检测技术领域，能够提高内部器件之间的固定稳定性，进而提高仪器的检测准确性和使用寿命。包括一侧铰接的底座和盖板，在底座上设置有待测标本固定架，以及围绕待测标本固定架设置的弹性部件、温控部件和通风部件，盖板上设置有光学模组，光学模组的光学扫描通道朝向待测标本固定架。实时荧光定量聚合酶链反应仪还包括开关组件，开关组件包括相互配合卡合的第一开关部件和第二开关部件，第一开关部件设置在底座的一侧，第二开关部件设置在盖板的一侧。