实时定量PCR监测B细胞恶性肿瘤患者造血干细胞移植后的IgH水平及意义

本研究评价实时定量PCR(RQ-PCR)技术检测IgH水平对B细胞恶性肿瘤患者造血干细胞移植(HSCT)后残留肿瘤细胞监测的意义.采用家族一致性TaqMan探针联合等位基因特异性寡核苷酸(ASO)上游引物技术检测22例B细胞恶性肿瘤患者HSCT前后骨髓单个核细胞的IgH水平动态变化.IgH水平以内参基因GAPDH进行归一化.结果表明,RQ-PCR实验可重复灵敏度为1个拷贝.9例IgH单克隆重排患者,在HSCT后1个月骨髓中IgH的拷贝数较初治时明显降低(6.67×103/106 GAPDH vs 29/106 GAPDH,p＜0.01).3例移植后15个月IgH的拷贝数持续小于102/106 GAPDH,18个月后IgH水平为0的患者获得了完全的临床和分子遗传学缓解(CCyR);5例移植后3个月以内IgH拷贝数持续小于102/106 GAPDH,随后IgH拷贝数持续小于103/106 GAPDH的患者临床获得完全缓解(CR).1例患者移植后近3个月时IgH拷贝数为4.5×103/106 GAPDH,4个月时临床复发.RQ-PCR检测8例干细胞采集物的肿瘤污染水平为3.68×102(0-1720)/106 GAPDH,外周血采集物中的肿瘤污染小于骨髓[75(0-890)/106 GAPDH vs 1.1×103(527-1720)/106 GAPDH,p＜0.05].采集物可用于RQ-PCR检测的8例患者无论肿瘤污染程度如何,临床均无复发.采集物中肿瘤污染的水平与初治及移植后1个月的IgH水平呈正相关(r值分别为0.810、0.708,p＜0.05).结论RQ PCR能够有效监测B细胞恶性肿瘤患者移植后IgH水平动态变化.移植后3个月内IgH拷贝数大于103/106 GAPDH可能是预测患者复发的标志.     

poly(A)加尾的实时荧光定量PCR检测血清miR-124a表达及临床初步应用

目的建立ploy(A)聚合酶加尾的SYBR Green I实时荧光定量PCR方法检测血清微小RNA(miR)-124a,并作临床初步应用。方法用Trizol试剂提取血清总RNA。miR-124aploy(A)聚合酶加尾逆转录获得cDNA,进行SYBR Green I实时定量PCR扩增检测。制作C.elegans-miR-39模拟物浓度梯度稀释的标准曲线,定量血清中miR-124a的表达水平。结果该方法能定量检测血清miR-124a表达水平,熔解曲线呈单峰,PCR扩增产物特异,在103~106 copies/uL范围内有良好的线性关系(r2=0.999),并且检测重复性好。糖尿病患者血清中miR-124a表达水平明显高于健康体检者(P0.05)。结论所建立的ploy(A)聚合酶加尾SYBR Green I实时荧光定量PCR方法能敏感、特异地检测血清中miR-124a的表达水平,为下一步临床应用的研究奠定了方法学基础。     

实时荧光定量检测中引物二聚体的优化

实时定量检测中引物二聚体的存在会严重影响检测结果的准确性,本文提出多方面的优化策略,以减少或消除实时定量检测中引物二聚体的集聚。     

SYBR Green Ⅰ荧光定量PCR检测宫颈癌血清中miR-21的表达

摘要：目的：建立SYBR GreenⅠ实时荧光定量PCR法检测宫颈疾病患者血清中miR-21的表达水平,探讨miR-21在宫颈癌临床监测中的意义。 方法：设计miR-21、U6茎环逆转录引物和PCR扩增引物,以U6为内参,用实时荧光定量PCR检测32例不同宫颈疾病患者血清中miR-21表达水平。 结果：建立的实时荧光定量PCR法能特异地检测血清中的miR-21扩增信号,熔解曲线单一,产物特异。宫颈癌患者血清中miR-21水平明显高于子宫肌瘤、宫颈炎等其他良性宫颈疾病（P<0.05）,而宫颈癌患者手术切除后血清中miR-21水平明显下降（P<0.05）。 结论：SYBR GreenⅠ实时荧光定量PCR是一种快速简便、敏感性高、特异性好的检测方法,对宫颈癌早期诊断、分子分期和预后判断有很好的应用前景。     

实时定量PCR法检测淋巴瘤患者Bcl-2／IgH融合基因及其临床意义

本研究目的是检测滤泡性淋巴瘤和弥漫大B细胞淋巴瘤患者bcl-2／IgH融合基因的表达水平,以探讨其临床意义。以SYBRGreenI荧光染料实时定量PCR方法检测20例患者外周血和（或）骨髓bcl-2／IgH融合基因的表达水平,并对其中4例患者bcl。2／IgH融合基因的表达水平进行动态监测。结果表明,在bcl-2／IgH融合基因阳性表达的病例中,骨髓和外周血bcl-2／IgH融合基因的相对拷贝数分别为4．23和2．73,统计学分析差异无显著性（P=0．107）,而治疗前后融合基因的相对拷贝数均数分别为3．61和2．69,统计学分析差异有显著性（P=0．000）,初诊及复发组的bcl-2／IgH融合基因表达水平明显高于缓解组（P=0．008）,在4例患者的外周血bcl-2／IgH融合基因动态监测结果中,l例患者在临床复发前3个月bcl-2／IgH融合基因表达水平明显上升。结论：bcl-2／IgH融合基因表达水平与患者的疾病状态有一定的相关性,初诊及复发患者融合基因的表达水平高,而缓解患者融合基因的表达水平低,治疗后融合基因表达水平较治疗前明显下降,bcl／IgH融合基因表达水平的变化可能与临床疾病进程有关,检测外周血是比较可行的。     

SYBR Green实时定量PCR检测SDF-1α基因在乳腺癌中的表达

目的 探讨SDF-1α基因在人乳腺癌中的表达及其与肿瘤恶性程度之间的关系.方法 SYBR Green实时定量PCR方法定量检测SDF-1α mRNA在63例乳腺癌（TNM分期：I期9例,IIA期25例,IIB期13例,IIIA期16例）和20例正常乳腺组织中的表达;采用Kruskal-Wallis检验、Mann-Whitney检验等分析SDF-1α mRNA表达在不同临床参数间的表达差异.结果 乳腺癌中SDF-1α mRNA表达水平（1.41±1.18）低于正常乳腺组织（2.02±0.71）,P=0.031.SDF-1α mRNA在乳腺癌中表达下调与淋巴结转移数目（〉3个腋窝淋巴结转移）、ER表达情况（0,1＋,2＋）密切相关（P值分别为0.017、0.047）.但SDF-1α mRNA在不同年龄、绝经情况、组织学分级、病理类型、肿瘤大小以及其他免疫组化指标（PR、Her-2、p53和Ki-67）组间的差异均无统计学意义（P〉0.05）.结论 趋化因子SDF-1α表达下调参与乳腺癌的转移,有望成为乳腺癌治疗的新靶点.     

SYBR Green Ⅰ荧光定量PCR检测类风湿关节炎患者血清miR-146a的表达及意义

目的用SYBR GreenⅠ实时荧光定量PCR法检测类风湿关节炎(RA)患者血清miR-146a的表达水平,探讨其在RA诊断中的意义。方法以U6为内参照,用SYBR GreenⅠ实时荧光定量PCR检测RA患者48例(活动期30例、缓解期18例)、骨性关节炎(OA)患者22例和体检健康者25例血清miR-146a的表达水平,并进行ROC曲线分析;对各组进行红细胞沉降率(ESR)检测。结果 SYBR GreenⅠ实时荧光定量PCR检测结果表明,活动期RA患者血清miR-146a水平为(2.42±0.75),显著高于缓解期RA患者(1.66±0.29)、OA患者(1.12±0.43)和体检健康者(1.01±0.34),差异均有统计学意义(P均0.05);miR-146a诊断活动期RA的ROC曲线下面积(AUCROC)为0.914(95%CI:0.872~0.926);以1.96为cut off值,敏感性为95.0%、特异性为87.5%、准确性为91.9%。活动期RA组ESR为(37.5±5.2)mm/h,缓解期RA组为(14.4±4.7)mm/h,OA组为(11.6±5.1)mm/h,健康对照组为(9.6±3.9)mm/h,各组间差异有统计学意义(F=177.01,P0.01)。结论建立的SYBR GreenⅠ实时荧光定量PCR可用于RA患者血清miR-146a的检测。miR-146a可用于诊断活动期RA。     

实时荧光定量PCR检测血清miR-375表达及临床应用

目的建立ploy(A)聚合酶加尾的SYBR GreenⅠ实时荧光定量聚合酶链反应(PCR)检测血清miR-375,并进行临床初步应用。方法用Trizol试剂提取血清总RNA。miR-375ploy(A)聚合酶加尾逆转录获得cDNA,进行SYBR GreenⅠ实时定量PCR扩增检测。制作C.elegans-miR-39模拟物浓度梯度稀释的标准曲线,绝对定量血清中miR-375的表达水平。结果该方法能定量检测血清miR-375表达水平,熔解曲线呈单峰,PCR扩增产物特异,在103~106 copy/μL范围内有良好的线性关系(r2=0.997),并且检测重复性好。糖尿病患者血清中miR-375表达水平明显高于健康体检者,差异有统计学意义(P0.05)。结论所建立的ploy(A)聚合酶加尾SYBR GreenⅠ实时荧光定量PCR能敏感、特异地检测血清中miR-375的表达水平,为下一步临床应用研究奠定了方法学基础。     

实时定量聚合酶链反应检测石蜡包埋B细胞淋巴瘤组织克隆性IgH基因重排

我们利用SYBR Green Ⅰ荧光染料，应用实时定量PCR（RQ-PCR）检测了15例B细胞非霍奇金淋巴瘤（B-NHL）患者免疫球蛋白重链（IgH）基因重排情况，以探讨克隆性IgH基因重排在B-NHL诊断和鉴别诊断上的价值。     

多重PCR检测急性淋巴细胞白血病IgH及TCRγ链重排基因

多重ＰＣＲ检测急性淋巴细胞白血病ＩｇＨ及ＴＣＲγ链重排基因徐兵周淑芸孙竞免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体（ＴＣＲ）基因重排，可作为淋巴细胞白血病特异性基因标志。应用多聚酶链（ＰＣＲ）技术检测重排基因以其灵敏、快速等特点而优于其它传统方法。但目前Ｐ...     

实时荧光定量聚合酶链反应检测大肠癌生存素基因

目的建立实时荧光定量聚合酶链反应(RT-PCR)法检测大肠癌组织生存素(survivin)mRNA的表达。方法用Primer5软件设计RT-PCR引物和TaqMan探针,建立survivin基因RT-PCR法;再用该法检测15例肠道良性疾病组和51例大肠癌及癌旁组织的survivin mRNA表达,以良性疾病组作为正常对照组。结果典型的阳性扩增曲线呈S形,无S形扩增曲线者为阴性,阳性标准质粒标准曲线线性好(r=0.99),正常对照组无survivin表达,大肠癌和癌旁组织的阳性表达率分别为88.24%和33.33%(P0.01),表达量分别是5.24±2.21和3.17±1.02(P0.05)。结论成功构建实时荧光定量RT-PCR法检测survivin基因;大肠癌组织中survivin高表达,表达与性别、肿瘤大小、Dukes分期及淋巴结转移无关(P0.05),正常对照组不表达;RT-PCR法是一简便可行、灵敏的方法,在大肠癌的普查及诊断治疗中有很高的应用价值。     

Detection of bcl-2/IgH rearrangements by quantitative-competitive PCR and capillary electrophoresis.

BACKGROUND: PCR is the primary method for detecting minimal residual disease in hematologic cancers. One such gene target is the bcl-2/immunoglobulin heavy chain (IgH) translocation found in a majority of cases of follicular lymphoma. METHODS AND RESULTS: We report an accurate method for quantitative detection of the bcl-2/IgH translocation marker of follicular lymphoma in a series of patients in various stages of remission and relapse who had been treated with a combination of ifosfamide, mitoxantrone, and etoposide (MINE) chemotherapy and monoclonal anti-CD20 antibody (Rituximab). The approach uses seminested PCR followed by analysis of the products on a fluorescent capillary electrophoresis system. The quantitation of bcl-2/IgH translocation-positive cells was sensitive and reproducible, capable of detecting as few as five malignant cells out of 300,000 normal cells. CONCLUSION: Quantitative PCR enables one to monitor the kinetics of tumor reduction in patients treated with MINE chemotherapy in combination with Rituximab.     

Detection and monitoring of minimal residual disease by quantitative real-time PCR

BACKGROUND: The detection of malignant cells by quantitative real-time PCR has become state of the art for diagnosis, monitoring response to treatment and detection of minimal residual disease (MRD) in patients with leukemia or lymphoma. In order to be used in high-throughput analyses technical details have to be standardized to improve reproducibility and comparability of quantitative results obtained in different laboratories. METHODS: Molecular monitoring of disease activity during and after treatment based on the detection of malignant cells in circulation or bone marrow by quantitative real-time PCR will be helpful to develop individualized treatment strategies for every patient. CONCLUSIONS: The effectiveness of any kind of innovative treatment with specific antibodies, cellular immunotherapy or molecules designed for specific targets of tumor cells can be controlled at a very high level of sensitivity and accuracy. Based on quantitative results indicative for success or treatment failure, therapeutic changes upon the detection of progressive disease at the molecular level can be made even before symptoms or signs of clinical relapse occur. Hopefully, this will lead to higher cure rates and improved long-term survival.     

自身免疫病患者增殖诱导配体基因mRNA表达水平的研究

目的建立检测增殖诱导配体(a proliferation-induc ing ligand,APR IL)mRNA的SYBR G reenⅠ实时定量PCR方法,了解系统性红斑狼疮(SLE)、类风湿性关节炎(RA)等自身免疫病患者外周血单个核细胞(PBMC)中APR IL的表达水平及其与自身免疫性疾病发病机制、预后之间的关系。方法构建克隆载体pGEM-T Easy-APR IL作为定量模板,定量检测58例自身免疫病(SLE、RA)确诊病人和20例健康献血者的外周血APR IL mRNA表达量,以APR IL mRNA和内参GAPDH mRNA表达量的比值作为APR IL表达水平的指标。结果自身免疫病患者的APR IL mRNA表达范围3.95~192.0,均值为29.68±4.5。20例正常对照者APR IL mRNA的表达范围3.1~18.7,均值为10.56±2.0。自身免疫病患者APR IL mRNA的表达水平高于正常对照组;久治不愈或疗效差者APR IL mRNA表达水平显著高于初发患者及治疗后缓解患者,但后两组差异无显著性意义。结论自身免疫性疾病(SLE、RA)患者的APR IL mRNA表达水平升高,疗效差、久治不愈患者表达水平更高。     

实时荧光定量PCR检测乳腺癌组织中KLK4 mRNA表达

目的探讨KLK4基因在人乳腺癌组织中表达的临床意义。方法用实时荧光定量PCR(FQ-PCR)的方法检测39例乳腺癌组织和25例正常组织中KLK4 mRNA的表达,以及mRNA表达量与雌激素受体(ER)、孕激素受体(PR)、CerbB-2及肿瘤的转移情况进行相关性分析。结果正常乳腺组织和癌组织KLK4 mRNA的相对表达水平分别为0.0120±0.0044和0.0272±0.0067,两者差异显著(P<0.01);乳腺癌组织中KLK4的相对表达水平在ER( )和ER(-)组分别为0.0269±0.0070和0.0276±0.0067;在PR( )和PR(-)组分别为0.0270±0.0065和0.0275±0.0081;在CerbB-2( )和CerbB-2(-)组分别为0.0270±0.0064和0.0301±0.0074;在肿瘤有、无转移组分别为0.0258±0.0061和0.0276±0.0066,各组间KLK4mRNA表达水平均无显著性差异(P>0.05)。结论在乳腺癌组织中KLK4 mRNA的表达水平显著高于正常乳腺组织,但在肿瘤是否转移,雌激素受体、孕激素受体及CerbB-2阳性和阴性组间无显著性差异。     

Basic principles of real-time quantitative PCR

Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.     

Detection of free-living amoebae by using multiplex quantitative PCR

Free-living amoebae (FLA) are protozoa found worldwide in soil and aquatic environments, which are able to colonize man-made water networks. Some FLA have the potential to be pathogenic and others might harbour pathogenic bacteria. Indeed, FLA feed on bacteria, but some bacteria could resist phagocytosis and either survive in FLA or even multiply within FLA. These bacteria are collectively named amoeba resistant bacteria (ARB). The best characterized example is Legionella pneumophila, for which FLA is the main reservoir in the environment. Not only could FLA be a reservoir that protects ARB, some bacteria might become more resistant to treatment and be more virulent. Thus, it is of medical significance to quantify FLA populations in soil, water or the environment. The main limitation for the quantification of FLA is that classical culture is not efficient and reliable for many genera and ‘strains’. Thus, several PCR-based quantification methods have been published for various FLA. However, thus far, no method has been published to simultaneously quantify the main FLA genera in the same PCR reaction. In this study, we developed a multiplex qPCR method to detect both Amoebozoan (i.e. Acanthamoeba, Hartmannella and Echinamoeba) and Vahlkampfiidae (i.e. Vahlkampfia and Naegleria) using 18S ribosomal RNA as the target gene. This method was shown to be specific, reliable and sensitive, could be used for the quantification of FLA and is likely to be useful to anticipate risks due to FLA or pathogenic bacteria, such as L. pneumophila.     

实时定量逆转录PCR在白血病标志物检测中的应用

目的 建立实时定量逆转录PCR（RQ-RT-PCR）检测各种急慢性白血病的特征性分子生物学标志物,评价其在疾病诊断和微小残留病（MRD）监测中的意义.方法 设计TaqMan探针和引物,建立RQ-RT-PCR法对各种融合基因转录本和ABL阳性模板进行扩增,并检测177份白血病标本的转录本含量,同时做细胞遗传学检查.结果 RQ-RT-PCR法最低可检测到10个拷贝/μl的阳性模板,但重复性较差,而（10^8～10^2）拷贝/μl的重复性良好,正常对照无扩增信号.与细胞遗传学相比,RQRT-PCR的敏感性和特异性更强,对白血病标志物的阳性检出率更高.对12例不同类型白血病患者的MRD随访监测表明：患者体内融合基因转录本的含量随病情进展逐渐升高,随病情好转逐渐下降,并且早于细胞遗传学预测疾病复发.结论 RQ-RT-PCR方法更敏感、可靠,与疾病的关系更密切,可用于白血病的诊断和MRD随访.