用于检测强直性脊柱炎相关基因HLA

1、用于检测强直性脊柱炎相关基因HLA-B27的实时荧光PCR芯片及其检测系统 ABSTRACT 概要The real-time fluorescent PCR chip and its detecting system used in examining the genes related to ankylosing spondylitis HLA-B27 ABSTRACT本文介绍了我们研制的一款实时荧光PCR芯片及其检测平台。我们将生物样品的预处理与 实时荧光PCR反应集成到一块芯片上。我们在芯片的腔体(2uL)内制作了一个坝，其下形成 了 1条1.5pm的罅缝，用于从血液中分离白细胞，然后在

2、同一腔体中进行PCR反应。相对 于常规管式PCR反应，本芯片能大大节省样品和试剂的使用量。利用微加工技术，我们还 将微加热器和温度传感器集成到本芯片上。芯片的封装采用硅和玻璃的阳极键合而成。针对 此芯片我们设计了相应的检测系统，它由注射泵模块、温度循环控制模块、荧光采集模块组 成。将此芯片和检测系统与我公司开发的EvaGreen HLA-B27 Detection Real Time qPCR Kit (Capitalbio)相结合，进行了强直性脊柱炎相关基因HLA-B27的检测。This article introduces a section of real-time fluorescen

3、t PCR chip and its testing platform developed by us. We integrated the biological samples pretreatment and the real-time fluorescent PCR reaction to one chip. We have built a dam in the chips cavity (2uL), and has formed a 1.5pm crack underneath it for separating the white blood cell from the blood,

4、 then carried out the PCR reaction in the identical cavity. This chip could save the amount of usage of samples and reagents compared with conventional tubular PCR reaction. Using micromachining technology, we also integrated micro-heater and temperature sensor on the chip. The material for the enca

5、psulation of the chip is anodic bonding of silicon and glass. We have designed the corresponding examination system in view of this chip, it is composed of injection pump module, temperature cycle control module and fluorescence gathering module. We have combined the chip and testing system with Eva

6、Green HLA-B27 Detection Real Time qPCR Kit ( Capitalbio)which developed by our company, and has carried out examination on the genes related to ankylosing spondylitis HLA-B27.INTRODUCTION 简介生物芯片技术是20世纪末发展起来的一项新技术。生物芯片是在微小面积上，利用微加工 技术，并结合有关的化学合成技术制造而成的一种具有一定分子生物学检验功能的微型器 件。分析和解释生物芯片上得到的信息，将在DNA结构与功能之

7、间架起一道桥梁，进而推 进生命科学的迅速发展。Biochip is a new technology developed at the end of 20 century. Biochip, the concept of it, is using the micro micromachining technology combines with the related chemical synthesis technology to fabricate a miniature component which bears certain molecular biology examination

8、 function. To analyze and interpret the information obtained from biochip will build a bridge between DNA structure and DNA function furthermore advance rapid development of life sciences.我们将生物芯片技术和实时荧光PCR技术相结合，设计了一款实时荧光PCR芯片。将样品 预处理和样品检测集成到同一块芯片上，能避免试剂污染，提高检测速度。We combined the biochip technology wi

9、th the real-time fluorescent PCR technology to have designed a section of real-time fluorescent PCR chip. And integrated sample pretreatment and sample detecting on the identical chip, so as to avoid the pollution of reagent and raise the detecting speed.芯片设计与制造Chip design and manufactureFigure 1(硅片

10、尺寸为 16mm*6mm，玻璃片尺寸为16mm*4mm，玻璃片上刻蚀出10mm*2mm的腔体)Figure 1 (The size of silicon chip is 16mm*6mm, glass sheet is 16mm*4mm and the size of the sculptured cavity on glass sheet is 10mm*2mm)(如图 1 所示)(as shown in Figure 1)芯片上用超声打孔技术打了 2个孔，用于试剂和生物样品的流进流出。Using supersonic drilling technique to drill 2 holes

11、on the chip, so as to let the reagent and the biological sample flow in and out.我们采用四端法测电阻检测测温电极，因此在芯片的设计中，测温电极上引出了四个端口(从 左往右数第2、3、6、7端口)。加热电极(共4根)对称排列于测温电极上下两侧，使芯片 上的温度能够均匀分布，再用较粗的电极将加热电极引出至芯片下端的外接端口(从左往右 数第 1、4、5、8 端口)。We apply the four-electrode method to measure the resistance and the temperatur

12、e measuring electrode, therefore in the design we have pinout four ports on the temperature measuring electrode (The 2nd, 3rd, 6th, 7 th from left to right). The heating electrode (total: 4) are arranged symmetrically in upper and lower sides of the temperature measuring electrode, so as to enable t

13、he temperature equably distributing on the chip, then use the thicker electrode to pinout the heating electrode on the external connection ports (The 1st, 4th, 5 th, 8th ports from left to right) which on lower side of the chip.当我们在硅片上刻蚀出所需的罅缝后，又在其表面制作了一层氮化曲二氧化硅/氮化硅层， 用作绝缘层。When we sculptured the re

14、quired crack on the silicon chip, we also produced a silicon nitride/silicon dioxide/silicon nitride layer on its surface serves as the insulating layer.芯片检测仪设计 Chip detection instrumentation design我们为此实时荧光PCR芯片设计了相应的检测控制系统。它具有注射泵模块、温度控制 模块和荧光采集模块。当我们从计算机输入整个系统的运行参数后，系统内的DSP将控制 并保证系统按要求运行。We have de

15、signed the corresponding detecting and control system for this real-time fluorescent PCR chip. It is composed of injection pump module, temperature control module and fluorescent gathering module. After we input the entire systems operational factors, the DSP in system will control and guarantee the

16、 system to operate according to requirement.在实时荧光PCR反应的应用中，需要用到多种荧光染料或荧光标记物，而不同的荧光染料 和荧光标记物需要不同波长的激发光。由于激光器发射光谱较窄，且价格昂贵，而不予采用。 我们采用白光LED作为激发光源，它的光谱范围在350nm800nm。对于特定的荧光染料， 通过选择相应波长的滤色片，就能满足它的激发要求。仪器中激发光滤色片和荧光滤色片设 计成可替换部件，在使用中我们能够根据使用的荧光染料特性挑选不同波长的滤色片。During the applying of real-time fluorescent PCR

17、reaction, it is necessary to use various kinds of fluorescent dyes or the fluorescent labels, but the different fluorescent dyes and the fluorescent labels need the exciting light with different wave length. Since the laser emission spectrum is a kind of narrow and the price is expensive, we are not

18、 put it into the practice. We use white light LED as the excitation light source; its spectral region is between 350nm to 800nm. Regarding the specific fluorescent dye, by choose the color filter with corresponding wave length to meet the request of excitation. The color filter of exciting light and

19、 fluorescent light in the instrument should be designed as replaceable components, in the usage, we can choose from various color filters with different wave length according to the characteristics of the using fluorescent dyes.To prevent fluorescence bleaching,我们只在PCR延伸阶段的最后几秒进行荧光的激发和采 样。非采样阶段，斩光器会

20、挡住LED发出的激发光，只有在荧光采集阶段，才打开斩光器， 使激发光照射到芯片上。To prevent fluorescence bleaching, we could only carry on the fluorescent exciting and sampling at the final several seconds of PCR extended period. During The non-sampling period, the photochopper would block the exciting light which LED sent out, only turn

21、on the photochopper in the period of fluorescence gathering, so as to the exciting light irradiate the chip.图2实时荧光PCR检测仪电路框图Figure 2 Electric circuit diagram for real-time fluorescent PCR detection instrumentation 我们使用两片具有低噪声、低输入电流偏移、低直流电压输出偏移特性的高精度运放OP27, 构成增益在1100可调的二级放大电路。We use two pieces of hig

22、h accuracy operational amplifiers OP27 characteristic of low noise, low offset for input current, low offset for DC voltage output, to constitute the second amplifying circuit and its adjustable gain is among 1 to 100.ADC采用了 Analog Devices公司的AD7665芯片，它支持最大010V的模拟电压输入和 16位数字信号输出，可以满足实时荧光PCR实验需要的动态范围和

23、灵敏度；ADC转换速度 最大为570kSPS，系统中由CPLD发出周期为16.5*的采样信号。这款芯片完全可以满足 转换速度的要求。We are using AD7665 of Analog Devices Corporation as ADC, the chip could be used for the maximal analogue voltage input among 0 to10V and 16-digital signal output, and meet the requirement of the real-time fluorescent PCR experiment f

24、or dynamic range and sensitivity; The maximal conversion rate of ADC is 570kSPS, the sampled signal is send out from the system by CPLD and the cycle is 16.5gs. This chip definitely could meet the request of conversion rate.DAC采用Analog Devices公司的10位D/A芯片AD5311，它采用I2C总线通信，方便了系 统布线，节省了系统所需的I/O端口，用于调节P

25、MT的增益。We are using the 10-digital D/A chip AD5311 of Analog Devices Corporation as DAC, it adopts the I2C bus communication to adjust the gain of PMT, which makes the system wiring more convenient and spares the required I/O ports.在此系统中，我们设计了 2个能独立工作的注射泵，分别用于血液样品和PCR混合溶液的 进样。In this system, we desig

26、ned 2 independent injection pumps for sampling injection of blood sample and the PCR mixed solution separately.CPLD (EMP7128SLC84, ALTERA)它外接一个 500KHz 的有源晶振，经过 CPLD 内部 5000 分频，向DSP提供1个100Hz的时基信号，用于控制AD采样和地址译码。DSP的地址线 全部引入CPLD中实现选址，数据线全部引入CPLD中实现读取数据。CPLD (EMP7128SLC84, ALTERA), an 500KHz external ac

27、tive crystal oscillator is connected with it, and divides the internal 5000 frequency through CPLD, then provides a 100Hz time-base signal to DSP for controlling the AD sampling and the address decoding. All address wires of DSP are led in CPLD to realize site selection, All data wires are led in CP

28、LD to realize data reading.图3 3块不同芯片测定的温度曲线Figure 3 Temperature curves for 3 different chips电极是用溅射的方式沉积到硅片表面，溅射的均匀性对芯片电阻影响很大。对每块芯片，我 们在50C、60C、70C、80C、90C这5个温度下测定它的阻值，标定出其阻值随温度变 化的曲线(如图3所示)。经过标定的测温电极具有0.1 C的检测精度。测温电路采用OP27 和AD620组成二级放大电路。AD620为低噪声仪用放大器，通过改变外接电阻的大小，可 以方便的调节其增益(11000)。经过放大后的信号引入DSP的10

29、位A/D。通过10次/秒 的温度采样，实现对PCR芯片的实时温度监控。The electrodes are deposited on the silicon chip surface with the sputtering method; the uniformity of sputtering is very important to the resistance of chips. For each chip, we determine its resistance under the temperature of 50 C, 60 C, 70 C, 80 C, 90 C, protrac

30、t the curves of changed resistance values consist with the temperature change (as shown in Figure 3). The detection precision of the protracted temperature measuring electrode is 0.1 C. The temperature measuring circuit adopts OP27 and AD620 to constitute the second amplifying circuit. AD620 is the

31、low-noise instrumentation amplifier; its gain (from 1 to 1000) can easily be adjusted through changing the value of external connection resistance. The enlarged signal is led in 10-digital A/D of DSP. Through temperature sampling which frequency is 10 times per second to realize the real-time temper

32、ature monitoring on PCR.控温模块由温度控制电路、芯片上的加热电极、降温风扇组成，用以实现PCR反应所需升 降温控制。加热电极的阻值为3Q左右，采用5V直流电压进行加热，最大升温速度大于20C /s。采用12V电压控制的风扇进行散热，最大降温速度大于8C/s。由于降温速度小于升温 速度，为了使系统升降温总时间缩短，我们将风扇设置为一直开启的状态。升温由DSP输 出的PWM信号控制加热电极实现，采用计数器Timer3。温度的控制程序采用PID算法实 现，通过对比例、积分、微分三个参数的调节，最终本系统温度控制精度达到0.2 Co Temperature controlled

33、 module is composed of temperature-control circuit, heating electrode on the chip and cooling fan, and it provides heating and cooling temperature control for carry out PCR reaction. The resistance value of heating electrodes about 3 Ohm, it adopts 5V DC voltage as power supply to carry on the heati

34、ng and the maximum heating rate is bigger than 20Celsius per second. And it adopts the fan administrated by 12V voltage for abstraction of heat; the maximum cooling rate is bigger than 8Celsius per second. Since the cooling rate is smaller than the heating rate, to cause the system to rise to decrea

35、se temperature the total time to reduce, we are about to set the fan active all the time to narrow the total time of heating and cooling. The heating is carried out by adopt Timer3 and output PWM signal control of DSP to control and heat the electrodes.The temperature control program adopts applicat

36、ion of PID algorithm, through the adjustment of three parameters (proportion, integral and differential) to finally make the precision of this system temperature control achieved 0.2Celsius.所有采集到的荧光数据和测温数据，都实时存储到计算机中，反应结束后，再在计算机上对 所采集到的数据进行分析处理。All gathered fluorescence data and temperature measurem

37、ent data are saved in computer in real time. After completed the reaction, we analyze and address the gathered data on the computer. 生物学应用Application on biology强直性脊柱炎是一种慢性发炎性的脊椎关节病变，好发病于年轻群体，这类疾病的众多症状 中，以病人的X光片影像学诊断最为重要。问题是病人从初始症状到发生X光影像明显变 化，常需时数年，因此，病人被误诊是非常常见的，据相关报道，男性病人平均被延误诊断 约5年，女性则为7年。因此，找到一种

38、更敏感有效的诊断方法是非常必要和迫切的。强 直性脊柱炎与HLA-B27高度相关，已被业内公认，通过研究已确定，强直性脊柱炎是一种 遗传性疾病，其患者的HLA-B27阳性率高达90%96%。目前，HLA-B27检测已成为临床 确诊强直性脊柱炎的重要手段之一。The ankylosing spondylitis is a kind of spondyloarthropathies and chronic inflammatory disease, it often occurs in the young community, among numerous symptoms of this kind

39、 of disease, the most important issue is the X-ray imaging techniques diagnosis for patients. The problem is, from the time of initial symptom has appeared to the time of X-ray imaging has obviously changed, it often takes several years, therefore, it is very common that the patient has been diagnos

40、ed wrong. According to the related report, the diagnosis of male patients is delayed approximately 5 years, in which the female is 7 years. Thus, find a certain kind of more sensitive and effective diagnosis method is very essential and urgent.The onset of this disease is highly related to the stren

41、gth of HLA-B27 and has been recognizedin the industry. Through the research we determined that the ankylosing spondylitis is a hereditary disease, the positive rate of its patients HLA-B27 reaches as high as from 90% to 96%, At present, the HLA-B27 examination has become one of the important means t

42、o make clinical accurate diagnosis of ankylosing spondylitis.我们将 CapitalBio EvaGreen HLA-B27 Detection Real Time qPCR Kit 与我们的芯片和检测仪 相结合，构成了一个快速检测HLA-B27的平台。此试剂盒基于荧光染料法测定PCR反应。 PCR反应产物通过熔解曲线进行进一步的分析。本体系包含两对引物一一质控引物、 HLA-B27扩增引物。如果样品只扩增出质控峰，则判定为阴性；如果样品扩增出双峰，则 判定为阳性。实际检测中为了结果的准确性，通过双峰的比值来判断阴阳性。We comb

43、ined CapitalBio EvaGreen HLA-B27 Detection Real Time qPCR Kit with ours chips and detecting instrumentation to constitute a rapid-test HLA-B27 platform. This kit is using the fluorescent dye method to measure the PCR reaction. The PCR reaction product carry on the further analysis by means of fuse c

44、urve. This system contains two pairs of primers 一一 quality control primer and HLA-B27 amplification primer. If the amplified sample only took sharp of quality control peak, then the sample is determined as negative; If the amplified sample took sharp double peaks, then it determined as positive. In

45、order to maintain accuracy of the result during the practical examination, we determine whether positive or negative it is by measure the proportion between the double peaks.配制20ML的PCR混合溶液备用：无菌蒸馏水(所有溶液均取自)取3gL血液样品用生理盐水将其稀释10倍，降低血液粘度。血液粘度太大将使得进样时需 加较大压力，使得白细胞变形，钻过坝底而到达另外一侧，达不到白细胞分离效果。Prepare 20 pL th

46、e PCR mixed solution: The sterile distilled water (all solutions are from)Take 3gL blood sample out and dilute it 10 times with the physiological salne to reduce the blood viscosity. If the blood viscosity too strong will cause the need of bigger pressure during the sample injection, so as to distor

47、t the white blood cell, drill through the bottom of dam to the other side, and failed to achieve the separation effect of the white blood cell.采用特制的机械卡具，使注射泵和芯片连接到一起，中间采用PEEK tubing(6007，Scienhome Science Instruments)进行连接。连接后体系的死体积小于1pL。稀释后的血液样品，用注射 泵以5gL/min的速度注入芯片。细胞分离效果如图4所示。然后将PCR混合溶液以2gL/min 的速

48、度进样，进样时间控制为1.5min，尽量赶走血液中的其他成分，而又不浪费太多的PCR 混合溶液。每次实验实际消耗PCR混合液为34pL。从芯片出口流出的废液用脱脂棉球收 集，样品进样结束后再用棉球将芯片出口擦干净。Adopting specialized mechanical fixture to connect injection pump with chip, adopt PEEK tubing (6007, Scienhome Science Instruments) as middle part for the connection. After the connecting, its

49、 dead volume is smaller than 1gL. We inject the diluted blood sample into chip with injection pump at the speed of 5gL per minute. And the cell separation effect as shown in Figure 4. Then injecting the sample at the speed of 2gL per minute with PCR mixed solution, the injecting time is limited at 1

50、.5min, in this way it expels the other ingredients in blood as far as possible and without waste the too many PCR mixed solution. The detecting actually consumed 3 to 4pL PCR intermixture at each time. The waste liquids which flow out from the exit of chip are gathered with the absorbent tampon, aft

51、er the sample injection has finished, use the tampon to wipe up exit of chip.图5细胞分离效果图Figure 5 The cell separation effect chartPCR扩增程序采用双温反应，即前10个循环采用较高的退火温度，保证产物的特异性，后 面的40个循环采用较低的退火温度，保证产量。反应温度控制方案如下表所示。实际温度 控制曲线图如图5所示(选取后40个循环中的一组控温数据)。The amplification procedure of PCR adopts dual temperature re

52、action, namely, during the first 10 circles maintain the higher annealing temperature to realize specificity of the products, and maintain the lower annealing temperature during the following 40 circles to realize the output. The reaction temperature control plan is showing as follows in the table.

53、The actual temperature control curve as shown in Figure 5 (selected the latter 40 circles as a set of temperature-control data).表1,反应温度控制方案Table 1, Reaction temperature control plan温 度(C)3796966872966172729642时间（s ）60090010151510151510循环数111040+0.1 C/s1Temperature(C)3796966872966172729642Time (s )60

54、090010151510151510CycleNumber111040+0.1 C/s1图5实际温度控制曲线图Figure 5 The curve for actual temperature control图6,实时荧光PCR扩增段数据分析(先起峰的为阴性样本，后起峰的为阳性样本) Figure 6, The data analysis for real-time fluorescent PCR amplification section (The first peak indicates the sample is negative and latter-coming peak indicates the sample is positive)图7实时荧光PCR熔解段数据分析(单