Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

APPLICATION OF GENETIC DEAFNESS GENE CHIP FOR DETECTION OF GENE MUTATION OF DEAFNESS IN PREGNANT WOMEN

Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom- ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 （35delG, 176del16bp, 235delC, 299delAT）, GJB3 （C538T） ,SLC26A4 （ IVS72A〉G, A2168G） and mito- chondrial DNA 12S rRNA （A1555G, C1494T） . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected, detection of deafness gene mutation carri- ers in 24 cases（4.2%）, including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took further genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta- tion has a high carrier rate in pregnant women group, 235delC and IVS7-2A〉G heterozygous mutations are common.

One Step Quick Detection of Cancer Cell Surface Marker by Integrated NiFe-based Magnetic Biosensing Cell Cultural Chip

RGD peptides has been used to detect cell surface integrin and direct clinical effective therapeutic drug selection. Herein we report that a quick one step detection of cell surface marker that was realized by a specially designed NiF e-based magnetic biosensing cell chip combined with functionalized magnetic nanoparticles. Magnetic nanoparticles with 20-30 nm in diameter were prepared by coprecipitation and modified with RGD-4C, and the resultant RGD-functionalized magnetic nanoparticles were used for targeting cancer cells cultured on the NiF e-based magnetic biosensing chip and distinguish the amount of cell surface receptor-integrin.Cell lines such as Calu3, Hela, A549, CaF br, HEK293 and HUVEC exhibiting different integrin expression were chosen as test samples. Calu3, Hela, HEK293 and HUVEC cells were successfully identified. This approach has advantages in the qualitative screening test. Compared with traditional method, it is fast, sensitive, low cost,easy-operative, and needs very little human intervention. The novel method has great potential in applications such as fast clinical cell surface marker detection, and diagnosis of early cancer, and can be easily extended to other biomedical applications based on molecular recognition.

Study on the Simultaneously Quantitative Detection for β-Lactoglobulin and Lactoferrin of Cow Milk by Using Protein Chip Technique

Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.

Simultaneous laser-induced fluorescence and contactless-conductivity detection for microfluidic chip

A combined detection system involving simultaneous LIF and contacfless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conduetivity detector was used in C^4D. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably.

THE STUDY ON NOVEL MICROELECTRODE ARRAY CHIPS FOR THE DETECTION OF HEAVY METALS IN WATER POLLUTION

Qualitative and quantitative analysis of trace heavy metals in aqueous environment are rapidly assuming significance along with the rapid development of industry.In this paper,gold microelectrode array(MEA)plated with mercury film was used for simultaneous voltammetric detection of zinc,cadmium,lead and copper ions in water.The electrochemical behavior and the actual surface area of the MEA were investigated by cyclic voltammetry in K_(3)[Fe(CN)_(6)].Electrochemical impedance spectrum(EIS)was utilized to examine the deposition of mercury on the electrode surface.Based on anodic stripping voltammetry,mercury film
Au MEA was applied to the detection of heavy metals in artificial analyte,where good calibrate linearity was obtained for cadmium,lead and copper ions,but with zinc exhibiting poor linearity.

An Integrated PDMS Electrophoresis Microchip with LED Induced Fluorescence Detection

A PDMS electrophoresis microchip,which integrated with optical fiber for fluorescence detection,was fabricated by using silicon master.A deep reactive ion etches (DRIE) technology was used to fabricate the silicon master with positive features.The PDMS replica was fabricated by casting PDMS prepolymer against the silicon master,where an optical fiber was first fixed on the end of separation microchannel.To improve the rigid characteristics of integrated PDMS microchip,the chips were subsequently assembled by reversible sealing against glass plate.A blue light emitting diode (LED) was used as excitation light sources for inducing fluorescence detection through coupling LED light into the optical fiber.As an application, integrated PDMS microchip was tested in the capillary electrophoresis separation of DNA markers.The results showed that DNA markers could be effectively separated and detected except for the segments of 271 and 281.

Combination of Micro-fluidic Chip with Fluorescence Correlation Spectroscopy for Single Molecule Detection

A single molecule detection technique was developed by the combination of a single channel poly （dimethylsiloxane）/glass micro-fluidic chip and fluorescence correlation spectroscopy （FCS）. This method was successfully used to determine the proportion of two model components in the mixture containing fluorescein and the rhodamine-green succinimidyl ester.

Research of Confocal Laser Induced Fluorescence Detection System for Micro-fluidic Chip

The characteristics such as signal noise ratio（SNR） and sensitivity of the fluorescence detection system for micro-fluidic chip influence the performance of the whole system extremely. The confocal laser induced fluorescence detection system is presented. Based on the debugging of optical and circuit modules, the results of detecting the samples are given and analyzed theoretically, and the improved project is put forward.

Development of a Liquid Chip Technique to Simultaneously Detect Taura Syndrome Virus( TSV) and Yellow Head Disease Virus( YHDV)

The aim is to develop a liquid chip technique to detect Taura syndrome virus( TSV) and yellow head disease virus( YHDV) on Penaeus orientalis simultaneously. The CP2 gene of TSV and N gene of YHDV in Gen Bank was analysed by using the software DNAStar 7. 0 to design the TSV-and YHDV-specific primers. The primers were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and then used for hybridization with RT- PCR products of TSV and YHDV. The liquid chip detection technique for detection of TSV and YHDV was established by using BD FACSArray to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to TSV and YHDV,with the detection of limit of 100 pg. Moreover,the assay was specific for the detection of TSV,YHDV and was not susceptible to cross with other viruses,including white spot syndrome virus( WSSV),spring viremia of carp virus( SVCV),infectious haematopoietic necrosis virus( IHNV). In conclusion,the liquid chip assay technique established in this study is highly sensitive and specific to TSV and YHDV detection. Moreover,it provides a novel,convenient and rapid approach for the detection of TSV and YHDV.

Sweat detection theory and fluid driven methods: A review

In recent years, analyses of sweat have become more popular since it doesn't require invasive sampling procedures. Although blood still remains the golden standards in clinical, analyses of other common body fluids,such as sweat, have become increasingly important. Because the compositions of sweat and blood are osmotically related, the content of certain metabolites in sweat can directly reflect the disease. Sweat detection can be used as an alternative to blood detection and allows continuous monitoring. Increased development of wearable sensors makes it possible for continuous sweat detection. Here, this paper gave a review about the sweat detection methods, such as fluorescence sensing, electrochemical sensing and colorimetric sensing. The advantages and disadvantages of each method and their developing trend in sweat detection were summarized. Then, for the problem of continuous sweat sampling, three methods(capillary force, hydrogel osmotic pump, evaporationdriven micropump) were introduced through different structures of microfluidic chip, and the level of sweat collection and transport achieved by related research was demonstrated. This review aims to provide guidance for future research in sweat detection and stimulate further interest in continuous monitoring of sweat using microfluidic chip.

A Simple and Reliable Assay for Detecting Specifi c Nucleotide Sequences in Plants Using Optical Thin-fi lm Biosensor Chips

Here we report the adaptation and optimization of an effi cient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-fi lm biosensor chips to detect unique transgenes in genetically modi-

Droplet microfluidic chip for precise monitoring of dynamic solution changes

In this work,an automated microfluidic chip that uses negative pressure to sample and analyze solutions with high temporal resolution was developed.The chip has a T-shaped channel for mixing the sample with a fluorescent indicator,a flow-focusing channel for generating droplets in oil,and a long storage channel for incubating and detecting the droplets.By monitoring the fluorescence intensity of the droplets,the device could detect changes in solution accurately over time.The chip can generate droplets at frequencies of up to 42 Hz with a mixing ratio of 1:1 and a temporal resolution of 3–6 s.It had excellent linearity in detecting fluorescein solution in the concentration range 1–5μM.This droplet microfluidic chip provides several advantages over traditional methods,including high temporal resolution,stable droplet generation,and faster flow rates.This approach could be applied to monitoring calcium ions with a dynamic range from 102 to 107 nM and a detection limit of 10 nM.

Screening differentially expressed genes from cyclin B1 down-regulated by high-throughput gene chip

Objective: To study differentially expressed genes by silencing cyclin B1, and to sift out autophagy-related genes. Methods: Double thymidine deoxyribonucleoside blocking was used to synchronize nasopharyngeal carcinoma cell (CNE-2) to S phase, then flow cytometry was applied to test transfection efficiency. The mRNA and protein expression level of cyclin B1 was assessed by q-PCR and western blot, respectively. Differentially expressed genes were screened by high-throughput gene chip. Results: Double thymidine deoxyribonucleoside (2.5 mmol/L) blocking was used to synchronize the cell cycle to S phase. The transfection efficiency of CNE-2 cells was 85.6%. Compared with negative group,cyclin B1-siRNA treated group significantly down-regulated mRNA expression of cyclin B1 (80%) and protein level (75.3%). Totally, 2408 differentially expressed genes were found in CNE-2, including 1245 up-regulated genes and 1163 down-regulated genes. Moreover, PTEN, an autophagy-related gene, was preliminarily sifted out. Conclusions: Cyclin B1-siRNA significantly down-regulated the expression of cyclin B1 and yielded a total of 2408 differentially expressed genes, including PETN (an autophagy-related gene).

A Customized Authentication Design for Traffic Hijacking Detection on Hardware-Trojan Infected NoCs

Traffic hijacking is a common attack perpetrated on networked systems, where attackers eavesdrop on user transactions, manipulate packet data, and divert traffic to illegitimate locations. Similar attacks can also be unleashed in a NoC (Network on Chip) based system where the NoC comes from a third-party vendor and can be engrafted with hardware Trojans. Unlike the attackers on a traditional network, those Trojans are usually small and have limited capacity. This paper targets such a hardware Trojan;Specifically, the Trojan aims to divert traffic packets to unauthorized locations on the NoC. To detect this kind of traffic hijacking, we propose an authentication scheme in which the source and destination addresses are tagged. We develop a custom design for the packet tagging and authentication such that the implementation costs can be greatly reduced. Our experiments on a set of applications show that on average the detection circuitry incurs about 3.37% overhead in area, 2.61% in power, and 0.097% in performance when compared to the baseline design.

用于土壤中氮钾含量快速测定的非接触电导微流控芯片

[目的/意义]土壤中氮、钾元素在作物生长和农业生产过程中具有关键作用。快速定量检测土壤中氮、钾含量对指导精确施肥具有重要意义。因此,建立一种快速可靠的土壤氮、钾含量检测方法十分必要。[方法]本研究建立一种基于聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)微流控芯片电泳和电容耦合非接触电导检测(Capacitively Coupled Contactless Conductivity Detection,C4D)方法,快速定量检测土壤中氮、钾养分离子。通过微流控电泳芯片实现对土壤中多种离子快速分离,利用C4D进行电导率变化的精准测量。基于检测器工作频率输出响应特性,激励电压响应特性和电泳电压,确定最佳分离和检测性能。[结果和讨论]该方法对钾离子(K+)、铵根离子(NH4+)和硝酸根离子(NO_(3)^(-))标准溶液的检测限(S/N=3)分别为0.5、0.1和0.4 mg/L。K^(+)、NH_(4)^(+)和NO_(3)^(-)在0.5~40.0 mg/L范围内具有良好的线性关系,线性相关系数(R2)分别为0.994、0.997和0.990,表明该方法可以对土壤中氮、钾养分离子进行定量分析。同时,采用峰高、峰面积和出峰时间作为评价指标进行可重复性实验,其相对标准偏差(Relative Standard Deviation,RSD)均小于4.4%,说明该方法具有良好的重复性。此外,对土壤样品进行测试,K^(+)和NH_(4)^(+)可实现完全分离以及同步检测,其检测效率明显提高。通过标准加入法进行回收率实验,回收率保持在81.74%~127.76%。[结论]本研究为土壤氮钾养分离子的快速检测提供了一种简便、高效的方法。

甲真菌病的真菌学检测方法研究进展

目前甲真菌病被认为是全球分布的主要公共卫生问题之一。甲真菌病的诊断依靠典型临床表现、真菌学检查(真菌镜检和真菌培养)以及甲病理检查。传统的真菌学检测方法在精准快速地鉴定真菌种类方面存在较大困难。免疫学诊断、基于PCR的分子鉴定、基因芯片技术、LAMP、MALDI-TOF MS及光谱学鉴定等新兴诊断技术具有快速、简便、精准的优点,能够克服传统诊断技术的局限性。本文阐述甲真菌病真菌学检测方法的研究进展,旨在发现和总结传统诊断方法与新兴诊断方法的优缺点,以便新兴检测方法的研究与发展。

基于RT-YOLO-V5的芯片外观缺陷检测

针对传统的人工芯片检测方法效率低、过分依赖人为操作且误检率高等产生的问题,提出了一种基于ResCBS模块与增加微检测层(Tiny-scale detection layer)的RT-YOLO-V5检测方法用于检测芯片外观缺陷。首先搭建了图像采集系统,并制作了芯片外观缺陷检测数据集。为解决芯片外观缺陷形状不规则、大小不统一、位置不确定带来的检测精度低等问题,在CBS模块中增加短连接,融合输入输出的特征信息,减少信息损失,优化推理速度;其次,增加一个微小尺度的检测层,提高模型对微小目标的特征提取能力。实验结果表明:使用改进后的网络对芯片外观缺陷进行检测,平均精度(mAP)达到95.5%,相对于原始网络提升了5.7%;除此之外,改进后的RT-YOLO-V5在先验框损失(Box_loss)与小目标缺陷的检测精度上都得到了一定的提升。

荧光纸基微流控芯片在食品检测领域中的应用

食品安全事故频发,引发全球对食品安全的广泛关注。为了保障食品的质量和安全,研究人员已经开发了各种快速和灵敏的检测方法,用于分析各种食品成分和污染物。纸基微流控芯片(μPADs)是以纸张为主要基版,使生化反应小型化的微实验室分析系统。近年来,μPADs在芯片设计和功能集成方面发展迅速,因其成本低、样品体积小和携带方便等优点,在快速检测中具有独特的应用潜力,已广泛用于保障食品安全。与其他检测方法相比,基于荧光法的μPADs具有选择性好和灵敏度高等优点,能够快速准确地测定目标物,满足现场检测的需求,为食品检测提供理想的解决方案。其在不同的实际应用中显示出广阔的前景,能测定食品中的各种污染物,包括病原微生物(大肠杆菌、金黄色葡萄球菌、沙门氏菌、单核细胞增生李斯特菌和副溶血性弧菌)、有害化学物质(农药、苏丹红染料、亚硝酸盐、甲醛和抗生素)和重金属离子(铅、镉、汞、砷、铬)等。笔者介绍了μPADs的基本概念,包括纸作为基材的优点以及二维和三维芯片的特点,重点介绍了荧光检测原理以及在食品检测领域中的应用。研究结果将有助于开发和应用更多新型基于荧光分析方法的μPADs,并为开发准确、灵敏、便捷的食品污染物检测方法提供参考。

基于STM32多传感器检测系统的设计与研究

为检测影响人们身体健康的生活环境状况,开发一种基于STM32单片机,以环境温度、湿度、可燃气体、有害气体等为检测对象,设计具有数据检测、显示及报警功能的设备。设备主要工作是通过多个传感器获取所在空间空气质量的参数信息,当参数浓度超过设定值时,单片机控制蜂鸣器报警,同时在OLED显示屏和手机用户端显示检测对象和当前浓度值,设计的目的是为人们的健康安全环境带来便利。