定量聚合酶链反应热循环系统及荧光检测技术研究
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摘要
聚合酶链反应(Polymerase Chain Reaction, PCR)是20世纪80年代中期发展起来的体外DNA快速扩增技术,在其基础上发明的定量PCR技术可以实现对反应物中DNA含量的准确定量计算,高性能的热循环系统和荧光检测系统是实现DNA扩增和定量的基础。本文对定量PCR中的热循环和荧光检测系统进行了研究：
热循环系统是DNA实现扩增的反应场所,本文设计了基于半导体制冷技术和嵌入式技术设计了PCR热循环系统。为了获得系统中各部件的准确参数,根据热电类比方法,建立了热循环系统的等效电路模型,利用该模型对热循环系统一维稳态和动态热性能进行了研究。结果表明该方法可以准确的对PCR热循环系统的一维热性能进行评估,并且可根据分析结果确定系统中各部件参数,使系统温度获得较好的稳态和动态性能。
PCR反应对温度要求较高,因此需要对热循环系统的温度控制方法进行研究,提高系统的温度控制精度。根据热循环系统的温度变化特点,提出了一种多模态分段控制策略,在升温和降温初期使用模糊控制方法,可以使系统获得最快的升降温速度,在升降温后期使用自适应模糊PID控制方法,可以有效提高温度稳态精度。实验表明,系统的最快升温速度大于4℃/s,最快降温速度大于2℃/s,稳态精度可以控制在0.2℃以内。通过实验得到了加热和制冷模式下,样品块到试液间温度传递时间与温度值关系,以保证PCR反应物有充分反应时间。
温度均匀性是系统中各样品等效率扩增的前提和保证。针对孔板式PCR普遍存在的温度场均匀性问题,利用有限元分析方法对影响系统温度均匀性的因素进行了研究,样品块表面与周围环境发生的热交换以及半导体制冷片之间的匹配误差是造成温度场均匀性差的主要原因。将半导体制冷片匹配误差控制在0.5%以内,再对热循环系统采取相应的隔热或热补偿措施,可以将系统温度均匀性控制在0.4℃以内。在此热循环系统中对标准PCR样品进行扩增实验,扩增产物的电泳成像结果表明系统的温度控制精度与均匀性可以满足PCR样品的高效扩增要求。
定量PCR通过检测反应体系中添加的荧光物质的荧光信号强度对反应物中DNA数量进行定量计算。根据荧光分析法原理和定量PCR反应过程中荧光染料的光谱特性,在基于共焦式荧光检测方法的基础上,设计了一种新型的定量PCR共焦荧光检测系统,使用光电倍增管和窄带滤光片对不同波长荧光信号进行检测。分析测试了荧光检测系统的噪声来源、降噪性能和荧光信号倍增效果,对采集到的荧光信号在计算机上进行数据段选取和数字滤波处理,最终计算出每个样品的荧光信号峰值。通过对不同浓度比的罗丹明B溶液以及添加了SYBR GreenⅠ染料的黄瓜基因组DNA溶液的荧光信号实际检测与处理,证明了系统可以检测最低浓度为0.005ug/ul的罗丹明B溶液发出的中心波长为610nm的荧光信号和0.78ng/ul的SYBR Green I溶液发出的中心波长为520nm的荧光信号,经过信号检测与处理后得到的信号峰值比基本与溶液的浓度比一致,可以满足定量PCR对指数期信号的检测与处理要求。
Polymerase chain reaction (PCR) is a technique used for rapid amplification of DNA in vitro. Quantitative PCR technology enables the accurate quantified calculation of DNAs in the reactant. High performance of thermal-cycler and fluorescence detection system is the basis of correct DNA amplification and quantitative. This dissertation carries on thermal-cycler and fluorescence detection method of a quantitative PCR system.
The thermal-cycler is a instrument which can implement the polymerase chain reaction, a PCR thermal-cycler was designed based on thermoelectric cooling technology and embedded technology. In order to get the accurate parameters of components in the thermal-cycler, the equivalent circuit model of thermal-cycler based on thermoelectric coolers was established according to the thermal-electric analogies and was applied to study the static and dynamic thermal performance of thermal-cycler in one-dimension. The results show that the method can accurately evaluate the thermal performance of PCR thermal-cycler in one-dimension and determine the parameters of components in the thermal-cycler. The system temperature can achieve excellent performances both in static and dynamic state.
PCR require precise reaction temperature. The temperature control method of the thermal-cycler system was studied in order to improve the control accuracy.fo the system temperature. According to the temperature characteristics of the thermal-cycler, a multi-modal segmented control strategy was studied. A fuzzy control method was used in the early stages of heating and cooling to get the fastest heating and cooling rate. An adaptive fuzzy-PID control method was used in the later period of heating and cooling to improve the static temperature precision. Experiment results show that the maxim heating rate of the system is greater than℃/s and the maxim cooling rate is greater than2℃/s, the static accuracy can be controlled at less than0.2℃. In order to ensure sufficient reaction times for the PCR samples, the relationship between hold-time and temperature was experimental studied in heating and cooling mode
The system temperature uniformity is a key factor to ensure the DNAs in various tubes can be amplified with equal efficiency. The factors affecting the system temperature uniformity were stuied by using finite element analysis method. The heat exchange between the surface of sample block and surrounding environment, and the matching error of thermoelectric cools are the main reasons for the poor temperature uniformity of the thermal-cycler. Experiment results show that the system temperature uniformity can be controlled in0.4℃by matching the thermoelectric cools with an error less than0.5%and implementing appropriate thermal insulation or compensation. The standard PCR samples were efficiently amplified in the thermal-cycler.
The DNAs in the samples are quantified by detecting the fluorescent intensity of fluorophores in each sample. A novel confocal fluorescence detection system of quantitative PCR was designed according to the principle of fluorescence analysis and the fluorescent spectra characteristics of fluorophores in the quantitative PCR process. Fluorescent signals with different wavelength can be detected by using a photomultiplier tube and different narrowband filters. Noise sources, denoise performance and the fluorescence signal multiplier effect of this fluorescence detection system were tested and analyzed. The peaks of the fluorescence signals of each sample were calculated on computer by segment-selecting and digital denoising the data acquired from the fluorescence detection system. Experiment results show that the system can detect the fluorescence signal (center wavelength of610nm) of rhodamine B solution with a lowest concentration of0.005ug/ul and the fluorescence signal (center wavelength of520nm) of SYBR Green I added to the cucumber genomic DNA solution with a lowest concentration of0.78ng/ul. The calculated peaks have linear relationship with the concentration and the system can detect the fluorescent intensity in the Exponential phase of quantitative PCR.
热循环系统是DNA实现扩增的反应场所,本文设计了基于半导体制冷技术和嵌入式技术设计了PCR热循环系统。为了获得系统中各部件的准确参数,根据热电类比方法,建立了热循环系统的等效电路模型,利用该模型对热循环系统一维稳态和动态热性能进行了研究。结果表明该方法可以准确的对PCR热循环系统的一维热性能进行评估,并且可根据分析结果确定系统中各部件参数,使系统温度获得较好的稳态和动态性能。
PCR反应对温度要求较高,因此需要对热循环系统的温度控制方法进行研究,提高系统的温度控制精度。根据热循环系统的温度变化特点,提出了一种多模态分段控制策略,在升温和降温初期使用模糊控制方法,可以使系统获得最快的升降温速度,在升降温后期使用自适应模糊PID控制方法,可以有效提高温度稳态精度。实验表明,系统的最快升温速度大于4℃/s,最快降温速度大于2℃/s,稳态精度可以控制在0.2℃以内。通过实验得到了加热和制冷模式下,样品块到试液间温度传递时间与温度值关系,以保证PCR反应物有充分反应时间。
温度均匀性是系统中各样品等效率扩增的前提和保证。针对孔板式PCR普遍存在的温度场均匀性问题,利用有限元分析方法对影响系统温度均匀性的因素进行了研究,样品块表面与周围环境发生的热交换以及半导体制冷片之间的匹配误差是造成温度场均匀性差的主要原因。将半导体制冷片匹配误差控制在0.5%以内,再对热循环系统采取相应的隔热或热补偿措施,可以将系统温度均匀性控制在0.4℃以内。在此热循环系统中对标准PCR样品进行扩增实验,扩增产物的电泳成像结果表明系统的温度控制精度与均匀性可以满足PCR样品的高效扩增要求。
定量PCR通过检测反应体系中添加的荧光物质的荧光信号强度对反应物中DNA数量进行定量计算。根据荧光分析法原理和定量PCR反应过程中荧光染料的光谱特性,在基于共焦式荧光检测方法的基础上,设计了一种新型的定量PCR共焦荧光检测系统,使用光电倍增管和窄带滤光片对不同波长荧光信号进行检测。分析测试了荧光检测系统的噪声来源、降噪性能和荧光信号倍增效果,对采集到的荧光信号在计算机上进行数据段选取和数字滤波处理,最终计算出每个样品的荧光信号峰值。通过对不同浓度比的罗丹明B溶液以及添加了SYBR GreenⅠ染料的黄瓜基因组DNA溶液的荧光信号实际检测与处理,证明了系统可以检测最低浓度为0.005ug/ul的罗丹明B溶液发出的中心波长为610nm的荧光信号和0.78ng/ul的SYBR Green I溶液发出的中心波长为520nm的荧光信号,经过信号检测与处理后得到的信号峰值比基本与溶液的浓度比一致,可以满足定量PCR对指数期信号的检测与处理要求。
Polymerase chain reaction (PCR) is a technique used for rapid amplification of DNA in vitro. Quantitative PCR technology enables the accurate quantified calculation of DNAs in the reactant. High performance of thermal-cycler and fluorescence detection system is the basis of correct DNA amplification and quantitative. This dissertation carries on thermal-cycler and fluorescence detection method of a quantitative PCR system.
The thermal-cycler is a instrument which can implement the polymerase chain reaction, a PCR thermal-cycler was designed based on thermoelectric cooling technology and embedded technology. In order to get the accurate parameters of components in the thermal-cycler, the equivalent circuit model of thermal-cycler based on thermoelectric coolers was established according to the thermal-electric analogies and was applied to study the static and dynamic thermal performance of thermal-cycler in one-dimension. The results show that the method can accurately evaluate the thermal performance of PCR thermal-cycler in one-dimension and determine the parameters of components in the thermal-cycler. The system temperature can achieve excellent performances both in static and dynamic state.
PCR require precise reaction temperature. The temperature control method of the thermal-cycler system was studied in order to improve the control accuracy.fo the system temperature. According to the temperature characteristics of the thermal-cycler, a multi-modal segmented control strategy was studied. A fuzzy control method was used in the early stages of heating and cooling to get the fastest heating and cooling rate. An adaptive fuzzy-PID control method was used in the later period of heating and cooling to improve the static temperature precision. Experiment results show that the maxim heating rate of the system is greater than℃/s and the maxim cooling rate is greater than2℃/s, the static accuracy can be controlled at less than0.2℃. In order to ensure sufficient reaction times for the PCR samples, the relationship between hold-time and temperature was experimental studied in heating and cooling mode
The system temperature uniformity is a key factor to ensure the DNAs in various tubes can be amplified with equal efficiency. The factors affecting the system temperature uniformity were stuied by using finite element analysis method. The heat exchange between the surface of sample block and surrounding environment, and the matching error of thermoelectric cools are the main reasons for the poor temperature uniformity of the thermal-cycler. Experiment results show that the system temperature uniformity can be controlled in0.4℃by matching the thermoelectric cools with an error less than0.5%and implementing appropriate thermal insulation or compensation. The standard PCR samples were efficiently amplified in the thermal-cycler.
The DNAs in the samples are quantified by detecting the fluorescent intensity of fluorophores in each sample. A novel confocal fluorescence detection system of quantitative PCR was designed according to the principle of fluorescence analysis and the fluorescent spectra characteristics of fluorophores in the quantitative PCR process. Fluorescent signals with different wavelength can be detected by using a photomultiplier tube and different narrowband filters. Noise sources, denoise performance and the fluorescence signal multiplier effect of this fluorescence detection system were tested and analyzed. The peaks of the fluorescence signals of each sample were calculated on computer by segment-selecting and digital denoising the data acquired from the fluorescence detection system. Experiment results show that the system can detect the fluorescence signal (center wavelength of610nm) of rhodamine B solution with a lowest concentration of0.005ug/ul and the fluorescence signal (center wavelength of520nm) of SYBR Green I added to the cucumber genomic DNA solution with a lowest concentration of0.78ng/ul. The calculated peaks have linear relationship with the concentration and the system can detect the fluorescent intensity in the Exponential phase of quantitative PCR.
引文
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