实时荧光定量PCR技术在临床肿瘤检测中的应用

肿瘤一直以来就是人类难以攻克的顽疾,近年来肿瘤疾病的爆发数量呈现上升趋势,如今我国肿瘤病例数约占全世界病例数的55%。实时荧光定量PCR技术(Real-Time PCR)是一种通过检测PCR产物中荧光讯号的强度从而进行定量的技术,这实现了对核酸信息量的分析比较。相对于常规的定性PCR技术,其具有检测范围宽、检测灵敏度高、精密度高、特性强、定量准确、重现性好等优点,现今已被广泛应用于生物学领域及医学领域尤其是肿瘤的早期诊断、分型、分期、预后诊断等。本文概述了实时荧光定量PCR技术的基本原理及其在肿瘤检测中的应用,并探讨了该技术的发展现状和应用前景。     

实时荧光定量PCR技术在动物遗传育种中的应用

聚合酶链式反应技术(polymerase chain reaction简称PCR)是一项体外基因快速扩增技术.该技术的建立,使定性检测的技术得到改善,达到检测单个靶细胞的水平,以其简便易行、灵敏度高等优点被广泛应用于分子生物学及医学等领域.但其易污染、假阳性及定量不准确等问题也日益突出.而荧光定量PCR技术(FQ-PCR)的出现解决了以上难题,实现了从定性到定量的飞跃.此方法特异性强和可靠性更强,能实现多重反应,且采用完全闭管检测,不需要PCR后处理,有效解决PCR污染问题以及EB对试验人员的伤害;再配以相应的荧光PCR仪,使整个PCR过程实现自动化,具有耗时短,操作方便等特点,因而广泛运用于各个领域.     

基于微流控荧光定量PCR的MRSA快速检测技术研究

针对耐甲氧西林金黄色葡萄球菌(MRSA)检测的问题,发展一种基于微流控荧光定量PCR的致病菌快速检测新技术。采用光刻法制作了微流控PCR芯片,研制了集成高性能温度控制模块和高灵敏度荧光检测模块的微流控荧光定量PCR分析系统。通过检测特异性基因femA和mecA对MRSA进行快速鉴别。实验结果表明,使用微流控PCR芯片可以成功实现MRSA特异性基因的快速检测,相对传统的管式PCR,芯片使用6μL试剂在56 min完成了MRSA特异基因的检测,不仅节约了反应试剂,而且极大提高了检测速度。该技术可扩展到其他致病菌的检测,具有良好的应用前景。     

光纤型定量PCR仪荧光检测光学系统性能研究

实践表明,光纤型定量PCR仪可以比共聚焦型定量PCR仪具有更高的荧光信号检测精度。然而,光纤型定量PCR荧光检测系统容易受到光源、透镜入瞳直径、光纤数值孔径NA和光纤损耗等诸多因素影响导致光源到光纤耦合效率下降从而影响系统的检测精度。针对光纤型定量PCR仪荧光检测光学系统,对其耦合效率进行Zemax仿真研究,根据仿真结果可以实现光纤型荧光检测光学系统的优化设计。     

荧光PCR多通道实时定量检测仪通过鉴定

6月21日，国家质检总局组织有关专家，对北京检验检疫局和深圳市匹基生物工程股份有限公司研制的荧光PCR多通道实时定量检测仪进行了鉴定。专家一致认为该仪器的研制成功，为荧光PCR检测技术在国内的推广应用提供了一种经济适用的检测工具，将在食品安全、兽医诊断、临床医学、生命科学研究等领域发挥重要作用。     

实时荧光PCR芯片及其检测平台的设计

研制了一款实时荧光PCR芯片及其检测平台.其中PCR芯片实现样品的预处理及实时荧光反应等功能;检测平台实现实时荧光检测、分析及处理等功能.该检测平台由注射泵系统、实时荧光信号采集系统和PCR温度控制系统等组成.并成功地实现了从血液中提取白细胞及同一腔体中PCR反应、检测以及分析等.实验结果表明,该系统能满足实时荧光PCR芯片的控制与检测实验结果,达到了设计各项指标.     

基于微流控芯片的数字PCR荧光图像分析方法

基于微流控芯片的数字PCR技术是当前痕量核酸分子绝对定量检测的先导技术,可通过荧光图像处理办法实现对目标分子高精度计量。本文基于本实验室大量数字PCR检测获取的荧光图像,开发了具备图像倾斜校正、图像二值化、芯片微反应腔室自动定位、图像快速拼接、图像分割、亮点计数等功能的荧光图像分析系统。该系统不仅实现了核酸分子的精确计数,且实现了较为友好的人机交互     

浅析食品快速检测技术在宾馆食品安全中的应用

对食品安全监测中实时荧光定量PCR技术与传统检测技术的区别和效用给以浅析,进而探讨PCR技术在宾馆食品安全关键控制点监测和公共卫生突发事件应急处理等方面的实际应用的可行性、必要性和发展前景。     

实时荧光定量PCR技术在水稻氮素基因表达中的应用之我见

实时荧光定量PCR技术实现了对低拷贝基因表达量的快速、准确定量,展望了荧光定量PCR技术在水稻氮素相关基因表达中的应用前景。     

便携式实时荧光定量PCR仪数据处理方法研究

实时荧光定量PCR越来越多被用来定量核酸的表达水平,随着经济与社会的发展进步,个人的自我保护意识和自身健康都重视了起来,使得人们对健康有了更高的要求,适应于现场核酸分析技术的需求日渐显著,不同的仪器有不同稳健的、合适的测量分析方法。本研究针对一种基于转盘式荧光检测的便携式实时荧光定量PCR仪的数据进行处理分析,通过对比优化不同峰值拟合算法及扩增曲线拟合算法优化提升了光学系统检测性能。结果表明,针对该系统峰值拟合算法采用正弦拟合对原始数据进行处理有较好的效果,扩增曲线拟合算法采用4参数logistic拟合,标准品浓度梯度样本扩增结果线性拟合优度R²>0.990,变异系数CV值小于5%,使得光学系统检测性能有所提升,为该类检测模型仪器的数据处理方法提供了一定参考。     

A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides

We develop a method, which utilizes a combined atomic force microscope (AFM)/fluorescence microscope and small copy number polymerase chain reaction (PCR), to affinity-select individual aptamer species in a single cycle from a small pool of random-sequence oligonucleotides (oligos). In this method, a library of small beads, each of which is functionalized with fluorescent oligos of different sequences, is created. This library of oligo-functionalized beads is flowed over immobilized target molecules on a glass cover slip. High-affinity target-specific aptamers bind tightly to the target for prolonged periods and resist subsequent washes, resulting in a strong fluorescence signal on the substrate surface. This signal is observed from underneath the sample via fluorescence microscopy. The AFM tip, situated above the sample, is then directed to the coordinates of the fluorescence signal and is used to capture a three-dimensional high-resolution image of the surface-bound bead and to extract the bead (plus attached oligo). The extracted oligo is PCR-amplified, sequenced, and may then be subjected to further biochemical analysis. Here, we describe the underlying principles of this method, the required microscopy instrumentation, and the results of proof-of-principle experiments. In these experiments, we selected aptamers in eight trials from a binary pool containing a 1:1 mixture of thrombin aptamer oligo and a nonsense oligo. In each of the eight trials, the positive control aptamer was successfully detected, imaged, extracted, and characterized by PCR amplification and sequencing. In no case was the nonsense oligo selected, indicating good selectivity at this early stage of technology development.     

Fluorescence in situ hybridization of three oncogenes on a leiomyoma

Using fluorescence in situ hybridization technique, expression of three oncogenes, C-myc, RARa, and cyclin-D was tested on a uterine leiomyoma. C-myc and RARa were amplified in approximately 30% and 90% of the cells, respectively. Numerous small signals of C-myc were indicative of the presence of double minutes. Amplification of RARa is being reported for the first time in a leiomyoma. Cyclin-D was normal in diploid cells while it was highly amplified in polyploid cells. Low levels of amplified C-myc and cyclin-D cells seem to be the reason for this tumor to be benign, while RARa could not be effective without the association of some other gene such as PML. Information presented here are significant toward developing new curative strategies such as gene-specific drugs and molecular manipulation to stop the activity of cancer gene. Further study may elucidate that how fibroids grow and maintain their rare benign nature.     

FEMTOLITER MICROARRAY WELLS FOR ULTRASENSITIVE DNA/mRNA DETECTION

ABSTRACT

DNA/mRNA analysis and genetic mutation detection are important in biomedical sciences, clinical diagnosis, and environmental monitoring. In this study, a femtoliter microwell array system has been developed for ultrasensitive DNA/mRNA detection using molecular beacon (MB) DNA probes. The microwell array system, with laser-induced fluorescence imaging, can be used to achieve a detection limit of 9 Rhodamin 6G molecules in 28?fL (28?×?10^?15?L) wells in this array. Hybridization kinetics of the MBs and their complementary DNA targets has been monitored with a concentration detection limit of 3.0?nM. As few as 50 target DNA copies can be monitored in each well. The specificity of MB and the effect of DNA target concentration on the MB hybridization kinetics have been investigated in femtoliter wells. Analysis of specific rat γ-actin mRNA sequence amplified by polymerase chain reaction (PCR) and simultaneous measurement of multiple analytes have also been studied with this microwell array system. As the volume in each well is extremely small, this array system will be highly useful for single cell gene profiling and for multiple gene determination in disease diagnosis.     

Double-pulse fluorescence lifetime measurements

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique (Müller et al. , 1995, Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc 177, 171–179).
A laser source with an optical parametric amplifier (OPA) system is used to obtain short pulse durations needed for high time resolution, wavelength tunability for selective excitation of specific fluorophores and high pulse energies to obtain (partial) saturation of the optical transition.
It is shown that fluorescence lifetimes can be determined correctly also with nonuniform saturation conditions over the observation area.
A correction scheme for the effect on the measurements of laser power fluctuations, which are inherently present in OPA systems, is presented. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique.
Because signal detection does not require fast electronics, the technique can be readily used for fluorescence lifetime imaging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.     

荧光光谱检测技术在茶饮料成分分析中的应用

荧光光谱检测技术已广泛用于有机物的定性和定量分析,取得了良好的效果.本文介绍了荧光光谱法的检测原理和检测系统的组成.采用该荧光检测系统在激发波长300～550nm、发射波长310～710nm处检测了茶饮料的荧光光谱,通过对荧光光谱的分析,建立了主要成分的荧光光谱特征数据库.该方法有助于解决市场上真假茶饮料的质量鉴别问题.     

基于PCA的时间分辨油荧光光谱分析及优化

激光诱导荧光技术可广泛应用于油污染的监测中,然而普通的油荧光光谱技术只能实现油污染监测的粗分类,无法区分原油与燃料油的荧光特征。本文基于主成分分析方法(PCA)的时间分辨油荧光分类方法,实验测量了20种油样本的时间分辨荧光光谱特征,给出了对应的荧光寿命和时间分辨油荧光光谱的时序特征。在此基础上,利用前三个主成分构成的三维特征矢量空间,通过分析不同采集时刻下油样本矢量间相关距离的变化,对油样本的时间分辨荧光光谱进行聚类分析。为了体现油荧光变化的时序性,引入矢量距离的离散度参量,提出基于PCA进行时间分辨油荧光光谱分析的优化方法。实验结果表明,基于时间分辨油荧光光谱识别可实现原油与燃料油的光谱时序特征区分,具备良好的油荧光分类效果。