人线粒体DNA荧光定量PCR检测方法的建立

建立SYBR Green I实时荧光PCR定量检测人线粒体DNA的方法。选取人线粒体DNA高度保守基因片段,将该基因片段与pCF-T载体连接后,转化入E.coli DH5α,提取重组质粒PCR及测序鉴定后,作为阳性模板建立SYBR-Green I荧光定量PCR标准曲线和熔解曲线。结果表明:构建的标准曲线线性关系良好(反应体系中含101~108拷贝时,扩增反应CT值与拷贝数的对数成线性关系),相关系数为0.997。批内和批间重复性测定的变异系数分别为1.23%~3.29%以及3.10%~5.21%。我们成功建立了实时荧光定量PCR检测人线粒体DNA的方法,该方法可作为进一步研究线粒体DNA的方法,在相关疾病诊断和监测中具有一定的应用前景。     

白纹伊蚊β-肌动蛋白实时定量PCR方法的建立与分析

采用T-A克隆技术,在白纹伊蚊β-肌动蛋白的基因序列上设计引物,PCR扩增后将特异性产物连入T载体中,提取质粒,经测序分析验证后,测定浓度,稀释获得标准品,采用SYBR green法进行实时定量PCR,并绘制标准曲线,作为白纹伊蚊各种基因实时定量PCR检测中的内参照物.结果表明：利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系（斜率为-3.3287,R2=0.9980）;实时定量PCR熔解曲线分析表明,温度在83℃±0.5℃的PCR产物是白纹伊蚊肌动蛋白序列上的特异性产物,表明此标准品是白纹伊蚊肌动蛋白特异性的.本实验成功建立白纹伊蚊β-actin基因实时定量PCR方法,可作为内参照物运用于白纹伊蚊基因差异表达的研究.     

应用荧光定量PCR检测缺失型DMD携带者的研究

目的建立荧光定量PCR技术检测缺失型DMD携带者.方法应用9对引物扩增抗肌萎缩蛋白缺失热点区域,检测出常见的缺失型DMD患儿17例.用SYBR Green Ⅰ荧光定量PCR技术对缺失型DMD患儿家系中21例女性亲属进行检测.结果1例肯定携带者、11例可疑携带者和3例可能携带者证实为携带者;5例可疑携带者和1例可能携带者被排除.结论SYBR Green Ⅰ荧光定量PCR技术是一种检测缺失型DMD携带者的准确、快速、简便的方法.     

SYBR green Ⅰ实时荧光PCR法检测KIR基因型

目的:建立一种新的杀伤细胞免疫球蛋白样受体(KIR)基因分型方法.方法:利用16对KIR序列特异性引物和两对内参照引物及荧光染料SYBR Green Ⅰ进行实时聚合酶链反应( real-time PCR),分析各扩增产物的熔解曲线,确定各个KIR基因熔解温度和特征,并以此为依据判断16种KIR基因的出现或缺失.对样本DNA进行不同倍数稀释检测该方法的敏感性.结果:分析熔解曲线能有效地进行KIR基因分型.该方法甚至可以对0.1 ng DNA的样本成功进行KIR分型.利用该方法成功地对10例外周血基因组DNA和10例宫颈细胞基因组DNA进行了KIR分型.结论:SYBR Green Ⅰ实时荧光PCR应用于KIR基因分型,具有简单、快速、敏感、实时和环保的特点,为实现KIR分型的自动化提供可能性.     

SYBR Green Ⅰ联合TaqMan荧光定量PCR检测HBV-DNA的意义

目的探索SYBR GreenⅠ联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为10^8.48、10^5.70和10^3.70copies/ml的3种HBV-DNA阳性血清和〈1×10^3.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green Ⅰ组成双荧光PCR（TaqMan＋SYBR Green Ⅰ组）,同时进行TaqMan和SYBR Green Ⅰ的单荧光PCR（分别为TaqMan组和SYBR Green Ⅰ组）,设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan＋SYBR Green Ⅰ组检测的HBV-DNA阳性血清均为阳性,其平均含量为10^8.55±0.32、10^5.79±0.29、10^3.81±0.30,与TaqMan组的10^8.49±0.31、10^5.69±0.34、10^3.72±0.26copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义（t=0.31、0.54和0.27,P〉0.05）;与SYBR Green I组的10^8.41±0.35,10^5.21±0.34和10^3.26±0.26copies/ml（不含未检出的两次血清）比较,除高浓度外,中低浓度有统计学意义（t=2.90和0.340.262.62,P〈0.05）。TaqMan＋SYBR Green I组和SYBR Green Ⅰ组阳性血清均出现明显熔解曲线,熔解温度（Tm）分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green Ⅰ联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。     

胃肠道恶性肿瘤C-myc基因荧光PCR方法的研究

目的建立胃肠癌中C-myc基因的荧光定量PCR(FQ-PCR)方法。方法胃肠癌细胞中的C-myc基因与质粒PGEM-T Easy Vector重组,转化大肠杆菌E.coli DH5α,获得克隆的C-myc基因标准模板。用ABI PRISM 7700PCR仪检测FQ-PCR扩增产物制成标准曲线来检测未知标本中C-myc的含量。结果 FQ-PCR扩增产物呈"S"形动力学曲线;ct(循环阈值)与PCR体系中起始模板拷贝数的对数值之间存在严格的线性关系,显示了FQ-PCR定量的准确性。结论 FQ-PCR是一种快速、简便、灵敏、准确的定量C-myc基因的方法。     

应用SYBR实时荧光定量Real-Time PCR法检测人宫颈癌MCPH1mRNA表达

目的 应用SYBR实时荧光定量RT-PCR法检测MCPH1/BRIT1 mRNA的表达.方法 提取人宫颈癌总RNA,经逆转录PCR获得靶基因(MCPH1)及管家基因(GAPDH)的CDNA,采用SYBR Green 荧光实时定量法检测,以GAPDH基因作为内参,计算各组MCPH1 mRNA的相对表达量.结果 在31...     

乳腺癌患者c-erBb-2基因荧光定量PCR方法的研究

目的： 建立乳腺癌中c-erbB-2基因的荧光定量PCR（FQ-PCR）方法。 方法： 乳腺癌细胞中的c-erbB-2基因与质粒PGEM-T easy vector重组，转化大肠杆菌E. coli DH5α，获得克隆的c-erbB-2基因标准模板。用ABI PRISM 7700 PCR仪检测FQ-PCR扩增产物制成标准曲线来检测未知标本中c-erbB-2的含量。 结果： FQ-PCR扩增产物呈“S”形动力学曲线；ct(循环阈值)与PCR体系中起始模板拷贝数的对数值之间存在严格的线性关系，显示了FQ-PCR定量的准确性。 结论： FQ-PCR是一种快速、简便、灵敏、准确的定量c-erbB-2基因的方法。     

BCR/ABL重组质粒实时荧光定量PCR标准品的制备

目的：用T-A克隆法构建含BCR/ABL融合基因的重组质粒,并用实时定量PCR（RQ-PCR）方法制备标准品。方法：通过培养细胞,提取总RNA并逆转录为cDNA后做PCR,电泳胶回收纯化,T-A克隆与pUCm-T载体连接,转染DH5a菌,蓝白斑筛选阳性菌落后,大量提取质粒,再进行RQ-PCR,最后制得BCR/ABL的重组质粒标准品。结果：蓝白斑筛选实验、PCR扩增均证实BCR/ABL融合基因重组到pUCm-T载体上,经RQ-PCR定量后得到BCR/ABL重组质粒标准品的标准曲线。结论：该方法能大量制备质粒标准品,并且可被推广应用。     

实时荧光定量PCR检测淋巴细胞中NKG2D、穿孔素和颗粒酶B基因的表达

目的:建立可用于测定淋巴细胞免疫功能的SYBRGreen I实时荧光定量PCR技术。方法:根据NCBI基因库中4种基因(NKG2D、穿孔素、颗粒酶B和内参照GAPDH)的序列,设计合成相应的引物,扩增上述基因。建立SYBRGreen I实时荧光定量PCR方法,检测肿瘤患者外周血淋巴细胞和诱导培养的患者CIK细胞(cytokine induced killer,CIK)中NKG2D、穿孔素和颗粒酶B mRNA的含量。结果:应用设计的引物扩增NKG2D、穿孔素和颗粒酶B基因后,经琼脂糖凝胶电泳和溶解曲线分析表明具有特异性。SYBR Green I实时荧光定量PCR检测结果表明,肿瘤患者的淋巴细胞中颗粒酶B基因的表达降低,而经细胞因子和单克隆抗体诱导培养的肿瘤患者CIK细胞与其淋巴细胞相比,细胞中穿孔素和颗粒酶B基因的表达明显增加(P0.01)。结论:该SYBRGreen I实时荧光定量PCR方法可用于检测淋巴细胞中NKG2D、穿孔素和颗粒酶B的mRNA的表达、作为研究淋巴细胞免疫功能的有力手段。     

Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.     

两种PCR方法检测狂犬病毒的敏感性和特异性比较

目的比较Nested-PCR和SYBR GreenⅠReal-Time PCR两种方法检测狂犬病毒的敏感性、特异性和时效性。方法对10倍连续稀释的狂犬病毒（疫苗株）核酸样品,采用Nested-PCR和SYBR GreenⅠReal-Time PCR方法进行平行检测,比较两种方法的敏感性;同时以登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸对两种方法的特异性进行评价。结果 SYBR GreenⅠReal-Time PCR方法可检测出2.3×106copies/μl核酸分子,与Nested-PCR方法相比,敏感度提高10倍。两种检测方法的特异性均好,与登革热2型病毒、诺如病毒、星状病毒、EV71和乙型脑炎病毒核酸均无交叉反应。SYBR GreenⅠReal-Time PCR方法检测花费3h,检测时间比Nested-PCR方法节省2h。结论与Nested-PCR相比,SYBR GreenⅠReal-Time PCR是相对高效的检测狂犬病毒的方法。     

Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma

PCR amplification and product analysis for the detection of chromosomal translocations such as bcl-1/JH have traditionally been performed as a two-step process with separate amplification and product detection. PCR product detection has generally entailed gel electrophoresis, hybridization, or sequencing for confirmation of assay specificity. By using a microvolume fluorimeter integrated with a thermal cycler and the PCR compatible double-stranded DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and detected bcl-1/JH translocation products by using rapid cycle PCR and fluorescence melting curve analysis. We analyzed DNA from 25 cases of lymphoproliferative disorders comprising 12 previously documented bcl-1/JH-positive mantle cell lymphomas, and 13 reactive lymphadenopathies. The samples were coded and analyzed in a blind manner for the presence of bcl-1/JH translocations by fluorescence melting curve analysis. The results of fluorescence analysis were compared with those of conventional PCR and gel electrophoresis. All of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at about 86 degrees C by melting curve analysis. For easier visualization of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T). Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 40-fold. Our results indicate that nucleic acid amplification integrated with fluorescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of specific PCR product detection without electrophoresis or expensive fluorescently labeled probes makes this methodology attractive for studies in molecular pathology.     

Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis

Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and β-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at ~90°C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (−dF/dT) versus temperature (T) showed melting temperatures (T_m) at 88.85 ± 1.15°C. In addition, multiplex assays using both MBR/JH and β-globin primers yielded easily distinguishable fluorescence melting peaks at ~90°C and 81.2°C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method for the detection of MBR/JH translocations. The feasibility of specific PCR product detection without electrophoresis or utilization of expensive fluorescently labeled probes makes this method attractive for routine molecular diagnostics.     

Development of a real-time PCR for the identification of Bordetella pertussis and Bordetella parapertussis

This study describes a real-time PCR assay for the detection and identification of Bordetella pertussis and Bordetella parapertussis. The assay is based on amplification of a fragment from the repeat sequence regions IS481 and IS1001 found in B. pertussis and B. parapertussis, respectively, with subsequent species identification by melting curve analysis using SYBR Green chemistry. Discrimination between the two species was straightforward, as the corresponding melting points showed a significant difference of 7 degrees C. The assay was evaluated first with reference strains and retrospective human clinical samples, and then prospectively with 132 human clinical specimens received between March 2003 and December 2005. The assay allowed the rapid detection of 22 positive clinical samples, of which 15, including one fatal case, were not identified by standard culture techniques. The new assay was sensitive and specific, and can be implemented easily using any real-time PCR apparatus.     

肾移植后BK病毒感染者实时荧光定量PCR检测

背景：对于BK病毒感染、BK病毒相关性肾病的认识缺乏规范的实验室诊断程序和标准化的无创性检验方法。 目的：建立肾移植后患者尿液和外周血BK病毒感染负荷实时荧光定量PCR检测方法。 方法：针对BK病毒基因组,自主设计特异性引物BKV-F和BKV-R,Taqman荧光探针BKV-P,Taqman荧光探针BKV-P的5’端标记有荧光基团,在除5’端外的任意一个位置上标记有淬灭基团;然后处理待测样本,进行PCR反应。 结果与结论：将检测阳性的扩增产物进行基因测序,测序结果经BLAST比对后证实为BK病毒基因序列;利用上述方法对56份样本进行检测,其中,20份BK病毒血清样本及20份BK病毒尿液样本检测均为阳性,有S型扩增曲线。动态范围测定显示在103~1010 copies/mL之间标准曲线具有良好的相关性。5份健康人尿液样本,5份血液样本及6份临床常见的其他病原体血液样本检测均为阴性,无S型扩增曲线。结果表明该方法可进行定性、定量检测,特异性好,反应快速,一般30 min即可得到反应结果,并且成本低、假阳性少。     

建立单管双向荧光PCR方法检测NQO1基因609C/T多态性

目的 建立单管双向荧光PCR方法快速测定人NAD(P)H:醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]基因609C/T多态性.方法 以人NQO1基因中的609C/T位点,设计双向引物,优化反应条件,应用SYBR GreenⅠ双向荧光PCR扩增191份人基因组DNA标本,并通过对产物进行熔解曲线分析,根据产物Tm值进行等位基因单核苷酸多态性分型.对其中62份标本用经典的PCR-限制性片段长度多态方法进行基因型分型,验证结果准确性.结果 62份样本的单管双向荧光PCR方法的基因型结果与PCR-限制性片段长度多态法分型结果符合率100%.191份样本中,纯合野生型(CC)占28%,杂合型(CT)占50%,纯合突变型(TT)占22%.结论 单管双向荧光PCR方法检测NQO1基因609C/T多态性,操作简便、反应过程快速,结果直观,敏感性、准确性和稳定性好,适用于临床样本检测及流行病学调查研究.     

应用SYBR GreenⅠ实时荧光聚合酶链反应检测SpA患者人类白细胞抗原-B27

目的评价SYBR GreenⅠ实时荧光聚合酶链反应检测人类白细胞抗原-B27。方法对65例SpA患者和100例健康人群,分别以PCR-SSP和SYBR GreenⅠ荧光PCR检测外周血HLA-B27 DNA进行检测,比较两种检测方法。结果丳CR-SSP检测HLA-B27阳性样本呈现135bp和268两个条带,阴性标本仅有268bp一条条带。SYBR GreenⅠRT-PCR检测结果示,阳性标本的熔解曲线呈现HLA-B27的90.37℃和β-globin的86.71℃两个Tm峰,阴性标本仅有β-globin一个Tm峰;侾CR-SSP和SYBR GreenⅠRT-PCR结果符合率为100%;僑pA患者和健康人的HLA-B27阳性率分别为70.76%和6%。结论 SYBR GreenⅠRT-PCR是一种可靠、快速的检测HLA-B27方法。