24重荧光real-time PCR技术在食物中毒快速检测中的应用

目的:应用24重荧光real-time PCR检测技术快速筛检食物中毒病原菌,结合国家标准中的培养法探讨24重荧光real-time PCR检测技术符合性和应用价值。方法:采用高灵敏度、高特异性的24重荧光real-time PCR检测技术作为中毒病原菌的初筛方法,国标方法进行细菌分离培养,并对分离出的病原菌进行生化鉴定。结果:5份食物中毒患者肛拭子在增菌前检出4份霍乱弧菌核酸(非O1/非O139群,24重荧光real-time PCR),9份患者肛拭子在增菌后检出5株霍乱弧菌(非O1/非O139群,国标培养法),其中包含4份PCR技术初筛阳性样品,两个方法的符合率为80%。所有样本均未检出沙门氏菌、志贺菌、副溶血性弧菌、致泻性大肠埃希菌以及金黄色葡萄球菌。结论:应用24重荧光real-time PCR检测技术同时检测24种常见致病病原菌,能高效锁定中毒病原菌。将其与国标培养法相结合,对临床治疗和食物中毒快速处置能起到积极作用,值得应用和推广。

Comparison of delay compensation methods for real-time hybrid simulation using frequency-domain evaluation index

The delay compensation method plays an essential role in maintaining the stability and achieving accurate real-time hybrid simulation results. The effectiveness of various compensation methods in different test scenarios, however, needs to be quantitatively evaluated. In this study, four compensation methods （i.e., the polynomial extrapolation, the linear acceleration extrapolation, the inverse compensation and the adaptive inverse compensation） are selected and compared experimentally using a frequency evaluation index （FEI） method. The effectiveness of the FEI method is first verified through comparison with the discrete transfer fimction approach for compensation methods assuming constant delay. Incomparable advantage is further demonstrated for the FEI method when applied to adaptive compensation methods, where the discrete transfer function approach is difficult to implement. Both numerical simulation and laboratory tests with predefined displacements are conducted using sinusoidal signals and random signals as inputs. Findings from numerical simulation and experimental results demonstrate that the FEI method is an efficient and effective approach to compare the performance of different compensation methods, especially for those requiring adaptation of compensation parameters.

A Class of Real-Time Parallel Combined Methods ofDigital Simulation for Large Systems

In this paper, a class of real-time parallel combined methods (RTPCM) of the digital simulation for a partitioned large system is presented. By means of combination of the parallelism across the system with the parallelism across the method, stiff and non-stiff subsystems are solved in parallel on parallel computer by a parallel Rosenbrock method and a parallel RK method, respectively. Their construction, convergence and numerical stability are discussed, and the digitalsimulation experiments are conducted.

Class of modified parallel combined methods of real-time numerical simulation for a stiff system

A class of modified parallel combined methods of real-time numerical simulation are presented for a stiff dynamic system. By combining the parallelism across the system with the parallelism across the method, and relaxing the dependence of stage value computation on sampling time of input function, a class of modified real-time parallel combined methods are constructed. Stiff and nonstiff subsystems are solved in parallel on a parallel computer by a parallel Rosen-brock method and a parallel RK method, respectively. Their order conditions and convergences are discussed. The numerical simulation experiments show that this class of modified algorithms can get high speed and efficiency.

A Real-Time Transient Analysis of a Functionally Graded Material Plate Using Reduced-Basis Methods

Based on the hybrid numerical method (HNM) combining with a reduced-basis method (RBM), the real-time transient response of a functionally graded material (FGM) plates is obtained. The large eigenvalue problem in wavenumber domain has been solved through real-time off-line/on-line calculation. At off-line stage, a reduced-basis space is constructed in sample wavenumbers according to the solved eigenvalue problems. The matrices independent of parameters are projected onto the reduced-basis spaces. At on-line stage, the reduced eigenvalue problems of the arbitrary wavenumbers are built. Subsequently, the responses in wavenumber domain are obtained by the approximated eigen-pairs. Because of the application of RBM, the computational cost of transient displacement analysis of FGM plate is decreased significantly, while the accuracy of the solution and the physics of the structure are still retained. The efficiency and validity of the proposed method are demonstrated through a numerical example.

A Decision Support Framework for the Choice of Languages and Methods for the Design of Real Time Embedded Systems

The choice of methods or design languages is a crucial phase in the development of systems and software, also for real time and embedded systems. An open question that remains in the design of these types of systems is to build a method, or to choose one among those existing, capable to cover the life cycle of a project, and particularly the development phases. This article contributes to answer the question, by proposing an approach based on a multi-criteria comparative study, of few languages and methods dedicated to the design of real time and embedded systems. The underlying objective of this work is to present to designers a wide range of approaches, and elements that can guide their choices. In order to reach this goal, we propose different comparison criteria. Each criterion is divided into sub-criteria, so that the designers can refine their choices according to the qualities they prefer and wish to have in the method or language. We also define a rating scale which is used to assess the retained languages and methods. The scores obtained from this assessment are presented in tables, one table per criterion, followed by a summary table giving the overall scores. Graphics built from these tables are provided and intend to facilitate the judgement and thus the choice of the designers.

Real-time embedded software testing method based on extended finite state machine

The reliability of real-time embedded software directly determines the reliability of the whole real-time embedded sys- tem, and the effective software testing is an important way to ensure software quality and reliability. Based on the analysis of the characteristics of real-time embedded software, the formal method is introduced into the real-time embedded software testing field and the real-time extended finite state machine （RT-EFSM） model is studied firstly. Then, the time zone division method of real-time embedded system is presented and the definition and description methods of time-constrained transition equivalence class （timeCTEC） are presented. Furthermore, the approaches of the testing sequence and test case generation are put forward. Finally, the proposed method is applied to a typical avionics real- time embedded software testing practice and the examples of the timeCTEC, testing sequences and test cases are given. With the analysis of the testing result, the application verification shows that the proposed method can effectively describe the real-time embedded software state transition characteristics and real-time requirements and play the advantages of the formal methods in accuracy, effectiveness and the automation supporting. Combined with the testing platform, the real-time, closed loop and automated simulation testing for real-time embedded software can be realized effectively.

Novel Real-Time Seam Tracking Algorithm Based on Vector Angle and Least Square Method

Real-time seam tracking can improve welding quality and enhance welding efficiency during the welding process in automobile manufacturing.However,the teaching-playing welding process,an off-line seam tracking method,is still dominant in automobile industry,which is less flexible when welding objects or situation change.A novel real-time algorithm consisting of seam detection and generation is proposed to track seam.Using captured 3D points,space vectors were created between two adjacent points along each laser line and then a vector angle based algorithm was developed to detect target points on the seam.Least square method was used to fit target points to a welding trajectory for seam tracking.Furthermore,the real-time seam tracking process was simulated in MATLAB/Simulink.The trend of joint angles vs.time was logged and a comparison between the off-line and the proposed seam tracking algorithm was conducted.Results show that the proposed real-time seam tracking algorithm can work in a real-time scenario and have high accuracy in welding point positioning.

Real- Time Color Enhancement Method Used for Intelligent Mobile Terminals

In certain environments and under some conditions, the video images taken by the intelligent mobile video phones seem dark, and the colors are not bright or saturated enough.This paper presents an adaptive method to enhance the video image brightness visualization and the color performance depending on the certain hardware property and function parameters. The experimental results prove that this method can enhance the colors and the contrast of the video images, based on the estimated quality feature values of each frame, without using the extra Digital Signal Processor (DSP).

Real-time quantitative optical method to study temperature dependence of crack propagation process in colloidal photonic crystal film

A real-time quantitative optical method to characterize crack propagation in colloidal photonic crystal film（CPCF）is developed based on particle deformation models and previous real-time crack observations. The crack propagation process and temperature dependence of the crack propagation rate in CPCF are investigated. By this method, the crack propagation rate is found to slow down gradually to zero when cracks become more numerous and dense. Meanwhile, with the temperature increasing, the crack propagation rate constant decreases. The negative temperature dependence of the crack propagation rate is due to the increase of van der Waals attraction, which finally results in the decrease of resultant force. The findings provide new insight into the crack propagation process in CPCF.

Study on Rear-end Real-time Data Quality Control Method of Regional Automatic Weather Station

[Objective] The aim was to study the rear-end real-time data quality control method of regional automatic weather station. [Method] The basic content and steps of rear-end real-time data quality control of regional automatic weather station were introduced. Each element was treated with systematic quality control procedure. The existence of rear-end real time data of regional meteorological station in Guangxi was expounded. Combining with relevant elements and linear changes, improvement based on traditional quality control method was made. By dint of evaluation and relevant check of element, the quality of temperature and pressure was controlled. [Result] The method was optimized based on traditional quality control method, and it narrowed the effectiveness of real-time data quality control. The quality check of hourly precipitation applied relevant check of hourly minimum temperature, vertical consistency check of radar data, which can effectively improve the accuracy and credibility of hourly precipitation quality control. [Conclusion] The method was on trial for one year in the quality control of real-time data in the regional automatic meteorological station in Guangxi and had gained good outcome.

猪IL-4、IL-6和IL-10 SYBR Green Ⅰ real-time PCR检测方法的建立及应用

为探讨PRRSV感染后机体的体液免疫应答、从分子水平深入研究PRRSV的免疫机制,本研究针对Th2型细胞因子IL-4、IL-6、IL-10以及看家基因β-actin的基因序列分别设计一对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-4、IL-6、IL-10及β-actin的SYBR Green Ⅰ real-time PCR方法。该方法线性关系好,各种细胞因子及β-actin标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到1×101copies/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3%。应用所建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)感染仔猪外周血单个核细胞(PBMC)中IL-4、IL-6和IL-10mRNA的表达水平进行了检测。结果表明,本研究建立的real-timePCR检测方法灵敏度高、特异性强、重复性好,可以用于猪Th2型细胞因子的检测及定量分析。

鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用

针对鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)DNA解旋酶基因编码区序列设计特异性引物,利用PCR技术扩增出长度为1 446 bp的基因编码区片段,克隆到pMD19T载体上,构建重组质粒。经PCR鉴定与测序分析确认正确后,以10倍梯度稀释重组质粒,作为标准模板进行TaqMan real-time PCR扩增,制作标准曲线,建立了鲤疱疹病毒Ⅱ型的荧光定量PCR检测方法。检测结果显示,标准曲线的相关系数(R2)达到0.999 1,斜率为-3.412;对初始模板定量检测的范围为1×101～1×107copies/μL;特异性试验结果表明,该方法可特异性地检测出鲤疱疹病毒Ⅱ型,而对大鲵虹彩病毒(GSIV)、锦鲤疱疹病毒(KHV)以及空白对照无检测信号。取江苏射阳和宝应两地疑似患病鲫组织核酸作为模板进行荧光定量PCR,结果表明反应体系中的病毒量分别为6.89×104copies/μL和3.02×102copies/μL。本研究建立的鲤疱疹病毒Ⅱ型TaqMan实时荧光定量PCR方法灵敏度高、特异性强,对因鲤疱疹病毒Ⅱ感染引起的养殖鲫造血器官坏死症的诊断与病毒病原定量检测有重要意义。

一种5重real-time PCR筛查转基因水稻方法的建立

以转基因水稻中最常用的CaMV35S启动子、NOS终止子、Cry1Ab/Ac基因、HPT基因及SPS水稻内标基因为研究对象,利用5种不同的荧光信号(FAM、HEX、Taxas Red、Cy5、Cy5.5)进行多重实时聚合酶链式反应(realtime polymerase chain reaction,real-time PCR)检测方法的研究。通过引物组合筛选、反应体系优化、特异性测试、灵敏度测试、适用性测试等一系列实验,建立了5重real-time PCR方法,灵敏度可达0.032%。此方法具有灵敏度高、结果准确、通量大等优点,可实现水稻中转基因成分的快速、高效检测。

金黄色葡萄球菌基因表达的DNA扣除法Real-time RT PCR相对定量分析

【目的】金黄色葡萄球菌作为一种分布广泛的致病微生物和研究革兰氏阳性菌遗传背景的模式菌株,利用real-time RT PCR对相关毒素及调控基因进行表达定量分析,在生物、医学、食品检测等领域具有较大研究价值。【方法】对制备好的反转录(RT,含有cDNA和DNA)和非反转录(RT—,仅含DNA)样品进行Real-time PCR检测,根据经典(1+E)—△△Ct相对定量算法并结合PCR效率公式建立一种基因表达相对定量分析的DNA扣除法,将得到的Ct值转换为各样品含量,从RT样品中扣除RT—样品的量,无需DNaseⅠ酶解处理就可以去除DNA的影响,RT—样品的检测结果还可同时作为稳定的DNA内参。【结果】采用以上方法分析金黄色葡萄球菌肠毒素A基因(sea)、16S rRNA和RNAⅢ的表达情况,在含有葡萄糖的NB培养基中sea的相对转录水平随着葡萄糖浓度的增大而升高,RNAⅢ的相对转录水平随葡萄糖浓度的变化而产生小幅度的波动,16S rRNA在菌体生长初期时的表达量较为稳定;与绝对定量法比较,结果差异较小(均小于15%),且差异不显著(p>0.05)。【结论】这种基于DNA扣除法的Real-time RT PCR相对定量方法可以有效的对金黄色葡萄球菌的基因表达进行分析。

应用real-time PCR定量检测土壤中小麦纹枯病菌方法的建立

根据小麦纹枯病菌Rhizoctonia cerealis AG-D融合群ITS区的DNA序列设计特异性引物对WKF-8/WKR-8，对引物的特异性和灵敏性进行检测，建立并优化基于 SYBR GreenⅠ荧光染料法的 real -time PCR反应体系，绘制标准曲线。检测范围在1．0×10-3～10 ng/μl之间有良好的线性关系，相关系数R2为0．995，扩增效率为99．7％，灵敏度比常规PCR方法高100倍。结果表明，该方法具有快速、特异性强、敏感度高等特点。

Data Transfer Over the Internet for Real Time Applications

Efficient real time data exchange over the Internet plays a crucial role in the successful application of web-based systems. In this paper, a data transfer mechanism over the Internet is proposed for real time web based applications. The mechanism incorporates the eXtensible Markup Language （XML） and Hierarchical Data Format （HDF） to provide a flexible and efficient data format. Heterogeneous transfer data is classified into light and heavy data, which are stored using XML and HDF respectively; the HDF data format is then mapped to Java Document Object Model （JDOM） objects in XML in the Java environment. These JDOM data objects are sent across computer networks with the support of the Java Remote Method Invocation （RMI） data transfer infrastructure. Client＇s defined data priority levels are implemented in RMI, which guides a server to transfer data objects at different priorities. A remote monitoring system for an industrial reactor process simulator is used as a case study to illustrate the proposed data transfer mechanism.

Real-Time Update of Sequence Placement Logic for High Arch Dams Based on Evidence Weight Discount

Sequence placement logic plays a significant role in construction simulation of high arch dams and directly affects the simulation process and results. To establish a sequence logic for dam block placement, the construction scheme, real-time construction process, and random factors of the site all need to be considered in detail. There are few studies available currently that take all these factors into consideration. To address this problem, a real-time update of sequence placement logic for high arch dams based on evidence weight discount is proposed in this study. First, the subjective weight of the dam block sequence priority criteria is built using a consistent matrix method based on the construction scheme. Second, using evidence theory, dynamic objective weight of the priority criteria and basic probability assignment is built. Finally, using a weight self-adaptive adjustment method and comprehensive evidence discounting, the placing probabilities of different dam blocks are obtained. A case study indicates that this method can realize real-time update of sequence placement logic. © 2017, Tianjin University and Springer-Verlag Berlin Heidelberg.

Velocity plus displacement equivalent force control for real-time substructure testing

This paper employs a velocity plus displacement（V＋D）-based equivalent force control（EFC） method to solve the velocity/displacement difference equation in a real-time substructure test. This method uses type 2 feedback control loops to replace mathematical iteration to solve the nonlinear dynamic equation. A spectral radius analysis of the amplification matrix shows that the type 2 EFC-explicit, Newmark-β method has beneficial numerical characteristics for this method. Its stability limit of Ω = 2 remains unchanged regardless of the system damping because the velocity is achieved with very high accuracy during simulation. In contrast, the stability limits of the central difference method using direct velocity prediction and the EFC-average acceleration method with linear interpolation are shown to decrease with an increase in system damping. In fact, the EFC-average acceleration method is shown to change from unconditionally stable to conditionally stable. We also show that if an over-damped system with a damping ratio of 1.05 is considered, the stability limit is reduced to Ω =1.45. Finally, the results from an experiment with a single-degree-of-freedom structure installed with a magneto-rheological（MR） damper are presented. The results demonstrate that the proposed method is able to follow both displacement and velocity commands with moderate accuracy, resulting in improved test performance and accuracy for structures that are sensitive to both velocity and displacement inputs. Although the findings of the study are promising, additional test data and several further improvements will be required to draw general conclusions.

基于Hsp70基因的绵羊肺炎支原体TaqMan检测方法的建立及其遗传演化分析

热休克蛋白70 (heat shock protein 70,Hsp70)是绵羊肺炎支原体(Mycoplasma ovipneumoniae, Movi)的重要膜蛋白,是机体内高度保守的生物分子,但在种间差异大,可作为分子生物学检测的候选靶区。为建立基于Hsp70基因的Movi通用型TaqMan实时荧光定量PCR(qPCR)检测方法,并进一步了解其遗传变异情况,本研究基于GenBank中Movi的Hsp70基因特征,设计特异性的引物及探针,建立了基于Hsp70基因的绵羊肺炎支原体qPCR检测方法。应用建立的检测方法对88份山羊鼻拭子样品及43份疑似羊支原体性肺炎(Mycoplasmal pneumonia of sheep and goats, MPSG)病料进行检测。将检测结果为Movi阳性的肺组织样品进行分离鉴定,并对分离株Hsp70基因进行序列分析。结果显示,建立的qPCR检测方法其相关系数为1.00,扩增效率为96.0%,斜率为-3.411,Y轴截距为37.29。特异性强,与丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri, Mmc)、山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae, Mccp)、莱氏无胆甾原体(Acholeplasmalaidlawii, AL)、无乳支原体(Mycoplasma agalactiae, Maga)、伪结核棒状杆菌(Corynebacterium pseudotuberculosis, CP)、羊口疮病毒(orf virus, ORFV)、牛支原体(Mycoplasma bovis, Mb)等牛羊常见病原均无交叉反应;敏感性高,最低检测限为5.72 copies·μL^(-1);重复性优,组内变异系数和组间变异系数均小于1.00%。6株Movi分离株Hsp70基因全长均为1 818 bp,与其它Movi参考株核苷酸和氨基酸同源性分别为96.0%~99.4%和98.0%~100.0%;进一步分析发现,山羊源Movi均比绵羊源多1个N-糖基化位点。遗传演化分析表明,其均处于ClusterⅠA亚分支(均为山羊源)。综上,本研究建立了特异性强、敏感性高、重复性优的基于Hsp70基因的Movi的qPCR检测方法。序列分析发现,不同来源Movi的Hsp70基因核苷酸同源性高;遗传演化分析证实,Movi福建株与山羊源分离株遗传关系较近。本研究不仅为Movi临床诊断提供了技术支持,更为进一步了解Movi遗传演化规律提供参考。