应用SYBR Green I荧光定量RT-PCR测定糖尿病大鼠肾皮质TGF-β1表达水平

SYBR GreenI荧光定量RTPCR法检测正常、糖尿病和牛磺酸治疗组大鼠TGFβ1mRNA/βactinmRNA比值为(7.0±0.8)×10-3,(64.4±8.0)×10-3和(16.7±2.0)×10-3;终点法RTPCR检测比值分别为0.28±0.12,0.58±0.16和0.43±0.10。与终点法RTPCR相比,TGFβ1mRNASYBRGreenI荧光定量RTPCR实时检测法,方便快捷,敏感特异。     

人类Gfi1基因SYBR Green I实时荧光定量PCR检测方法的建立

目的 建立测定人类Gfi1mRNA表达量的SYBR Green I实时荧光定量PCR方法,为进一步研究新基因的功能奠定基础.方法 以Gfi1基因为靶基因设计引物.构建携带GficDNA的质粒plox-Gfi1,经过纯化,质粒浓度测定,拷贝数的计算及10倍的梯度稀释制备标准品.应用ABI stepone PCR仪对Cfi1mRNA进行实时荧光定量PCR检测,建立标准曲线和熔解曲线.结果 建立了Cfi1mRNA表达的实时荧光定量PCR方法,本法线性范围为6.36×102～6.36×108copies/μl,线性相关系数y2为0.996,熔解曲线为单峰,Tm值为(89.98±0.22)℃,标准品的循环阈值批内变异系数和批间变异系数分别为1.35%～3.61%和1.49%～3.42%.结论 本研究建立的Gfi1mRNA表达实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性及稳定性好,可用于Gfi1基因的定量检测.     

SYBR Green实时定量PCR检测胰腺癌中HIF1α、VEGF和bFGF的表达及其临床意义

目的:研究胰腺癌及癌旁组织中HIF-1α、VEGF和bFGFmRNA的表达情况及其临床意义。方法:利用实时荧光定量聚合酶链反应技术对22例胰腺癌细胞及其对应的癌旁组织中HIF-1α、VEGF和bFGFmRNA进行定量检测,并分析其与临床病理特点之间的关系。结果:统计结果显示HIF-1α、VEGF和bFGF在胰腺癌中的表达高于癌旁组织(P<0.01),相关性分析显示肿瘤组织中HIF-lα表达与VEGF、bFGF表达呈显著正相关(P<0.01)。HIF-1α表达与肿瘤大小和淋巴结转移显著相关(P<0.01),与TNM分期有关(P<0.05),与性别、年龄无相关性,VEGF表达与肿瘤大小和淋巴转移相关(P<0.05),bFGF高表达与肿瘤大小、淋巴转移淋巴转移相关(P<0.05),而与TNM分期、性别及年龄无相关性。结论:HIF-1α、VEGF和bFGF在胰腺癌中存在高表达,HIF-1α表达与TNM分期、肿瘤大小和淋巴转移相关,VEGF和bFGF表达与肿瘤大小和淋巴转移相关。实时荧光定量PCR检测HIF-1α、VEGF和bFGF对评估胰腺癌患者预后可能有一定的价值,也为胰腺癌的治疗提供了治疗靶点。     

SYBR Green实时定量PCR检测人原发脑胶质瘤中REV3和REV7基因的表达

目的 运用实时定量PCR检测跨损伤修复基因REV3和REV7在人脑原发脑胶质瘤组织中的表达水平,探讨其和肿瘤级别之间的关系.方法 采用SYBR Green实时定量聚合酶链式反应(RT-PCR)法检测跨损伤修复基因REV3和REV7的mRNA在85例原发胶质瘤(Ⅱ级20例、Ⅲ级20例和Ⅳ级45例)和14例正常脑组织中的表达水平,统计学分析表达水平和肿瘤级别之间的关系.结果 与正常组织相比,REV3和REV7在各病理级别胶质瘤中均表达上调(P＜0.05);并且REV3在Ⅳ级胶质瘤中的表达比在Ⅱ级和Ⅲ级中都要高(P＜0.05).秩相关分析表明:REV3的表达量与胶质瘤病理分级呈正相关性(r=0.454,P＜0.001).结论 REV3和REV7在胶质瘤组织中均高表达,而且REV3表达水平和恶性程度有密切联系.     

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C_t) 18.19 ± 3.63 and a melting temperature (T_m) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C_t value 14.70 ± 2.32 and T_m 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log₁₀ concentration range, 10⁹–10⁵ copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.     

Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

OBJECTIVE: To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique. MATERIAL AND METHODS: Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species. RESULTS: A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number. CONCLUSION: A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.     

实时定量RT-PCR方法检测豚鼠TSHR免疫BALB/c小鼠甲状腺TSHR的表达

目的：建立外标准法SYBR GREENI实时定量（real—time）RT—PCR方法，以检测豚鼠TSHR分子免疫对BALB／c小鼠甲状腺TSHRmRNA的表达的影响。方珐：取正常BALB／c小鼠甲状腺提取甲状腺总RNA．逆转录后进行PCR，得到333bp扩增产物。经回收纯化后作为real—time PCR的标准品。稀释为10^6、10^5、10^4、10^3、10^2、10^1拷贝，制作标准曲线；并同时进行普通PCR，比较其灵敏度。优化反应条件使溶解曲线有特异扩增。检测豚鼠TSHR分子免疫的BALB／c小鼠甲状腺TSHR mRNA的表达。结果：建立的外标准法SYBR GREENI实时定量RT—PCR方法可以灵敏的检测到10copy的核酸，灵敏度为普通PCR的10^4倍；溶解曲线只有特异峰，溶解温度为82．9℃。没有明显的引物二聚体和非特异峰出现。不同标准点批间变异系数范围为5．8％．14．3％（n=6）。与对照组比较，TSHR大分子免疫的BALB／c小鼠甲状腺组织TSHR mRNA水平显著升高（P〈0．05）。结论：外标准法SYBR GREENI实时定量lit—PCR具有灵敏度高，特异性强，重复性好的特点，比普通PCR更精确地检测出小鼠甲状腺组织TSHR mRNA的水平。     

实时荧光定量PCR检测转基因小鼠外源基因拷贝数方法的建立和应用

目的建立实时荧光定量PCR方法检测CaMKⅡα-Cre转基因小鼠外源基因拷贝数的方法。方法以CaMKⅡα-Cre转基因首建鼠及其仔代阳性鼠为研究对象,利用绝对定量的实时荧光定量PCR法检测转基因小鼠的拷贝数,并筛选出纯合子小鼠再经遗传育种方法以确定为纯合子小鼠。结果绝对定量标准曲线公式为:△Ct=-2.402log5N(拷贝数)+8.654,相关系数为0.9999,扩增效率为95.4%。三只CaMKⅡα-Cre首建鼠的拷贝数分别为19、7、5;三个转基因小鼠品系均成功获得纯合子小鼠。结论成功建立了检测转基因小鼠外源基因拷贝数的实时荧光定量PCR方法,该方法可用于检测各种转基因小鼠中外源基因的拷贝数。     

基于特异性聚合酶链式反应的西红花快速分子鉴别研究

 目的 建立一种基于特异性聚合酶链式反应(PCR)及荧光染料检测快速鉴别西红花真伪品的方法。方法 通过对叶绿体基因片段测序和比对分析,找出西红花与其常见掺伪品序列差异,针对差异碱基设计特异性引物,构建并优化聚合酶链式反应扩增反应体系,使用荧光染料法检测聚合酶链式反应产物,实现西红花的快速鉴别。结果 建立了基于psbA-trnH序列的西红花鉴别体系,优化后的聚合酶链式反应反应条件为90 ℃预变性1 min,90 ℃变性5 s,58 ℃延伸5 s,26个循环。在聚合酶链式反应反应产物中加入染料SYBR Green I,西红花正品出现强烈绿色荧光,而伪品无荧光。结论 位点特异性聚合酶链式反应方法可以简单快速鉴别西红花及其掺伪品。