寡核苷酸探针悬浮芯片在病原体检测中的应用

悬浮芯片与固体芯片、荧光定量PCR并列成为核酸序列鉴定中的重要的分子生物学工具,并在病原菌检测方面显示出不同的应用领域.悬浮芯片能同时检测多种病原菌,具有处理多样本能力、使用灵活、低成本等特点,适合对未知样本检测及环境监控.能够在生物安全、公共卫生、工农业生产中发挥重要作用;而固体芯片能耦联成千上百个探针,但由于在多样本处理、成本方面欠缺,因此适合于对重要的未知病原体的鉴定;荧光定量PCR具较好特异性、灵敏度,以及多样本处理能力,但在高通量方面欠缺.适合有目的地检测已知病原体.目前已建立三种基于悬浮芯片的检测方法:多重PCR扩增、通用引物扩增16S/23S rDNA、直接对实际样本杂交检测.多重PCR具较好特异性,但其多重能力还难以满足悬浮芯片的高通量的需要;通用引物具较好灵敏度及扩增多靶分子能力,但也存在交叉反应等缺陷.同时,采用PCR扩增方法,悬浮芯片检测的是PCR产物,不能客观反应实际样本中存在病原菌数量及是否具生命力.直接杂交环境样本尽管避免了PCR的缺陷,但在灵敏度方面非常欠缺.目前,在环境样本处理上,仍然缺乏有效的、高通量、自动化的方法,不能满足PCR与悬浮芯片多样本检测的需要.     

悬浮芯片在核酸和蛋白质检测中的应用

悬浮芯片是近年来兴起的一种新型检测技术,不同于固相基因芯片,它整合了高分子化学、分子生物学、免疫学、激光检测、微流体、高速数字信号处理、计算机分析等方面的先进技术,能够对少量样本进行高通量的定性、定量检测。主要综述了悬浮芯片技术的基本原理,并概要介绍了其在核酸和蛋白质检测中的应用。悬浮芯片技术在核酸和蛋白质检测中有着显著的优点,如高通量、操作简便、重复性好、灵敏度高、线性范围宽等,不但可以广泛应用于科学研究领域,而且还将逐渐普及于临床诊断实验室,具有广阔的应用前景。     

Analysis of Dual-Channel Simultaneous Detection of Photonic Crystal Fiber Sensors

Plasmonics - Photonic crystal fiber (PCF) sensor based on surface plasmon resonance (SPR) has broad application prospects in the detection of biological proteins, DNA/RNA, and toxic chemicals....     

高通量流式荧光微球悬液芯片检测乙肝病毒基因分型

目的:建立HBV基因分型高通量液相芯片检测技术,并探讨其应用价值.方法:对GenBank中收录的明确分型的HBV基因序列进行分析,选择preS2-S区设计引物和A、B、C和D型特异性探针.与荧光编码微球偶联的特异型探针与一条引物生物素标记的PCR产物直接杂交反应,然后结合亲和素标记的藻红蛋白,用流式检测仪(Bio-Plex 200)检测荧光信号.检测182份阳性乙肝患者血清DNA,其中35份样品检测结果与测序法比较.用B、C型质粒DNA倍比稀释及混合样品检测灵敏度来评估该方法.结果:建立了HBV基因分型的快速高通量液相芯片检测方法.182份患者血清检测结果为:B型占24.2％ (44/182),C型占71.4％(130/182),D型为6.6 ％(12/182),BC混合型4.4％(8/182).其中35份样本与测序法比较,除3份混合型测序法未检出外,其它32例结果均相同本方法的灵敏度检测下线为1×103 copies/mL.结论:应用悬液芯片技术进行乙肝病毒的基因分型分析,具有较好的特异性和较高的灵敏度,并有简便、灵活和高通量等优势.该检测系统不仅在科研中有广泛的前景,也有望成为临床推广的多重分子诊断和基因分型的新方法.     

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.     

The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array

Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.     

悬浮芯片技术应用进展

悬浮芯片技术(SAT)是一种新型、高通量的生物芯片技术,它是将流式细胞术、激光技术及应用流体学等技术结合在一起,利用悬浮在液相中的分类荧光编码微球作为检测载体,具有高通量、速度快、灵敏度高、特异性强及检测范围广等特点.近几年来,悬浮芯片技术在免疫学、基因组学、蛋白质组学及临床诊断检测等方面应用较广泛.就其原理、技术特点...     

Observation of an Optical Bound State in Photonic Crystal Slabs with U-Shape Nanohole Array

Plasmonics - We have theoretically investigated the reflectance spectrums of the U-shape nanohole symmetry and asymmetric structure. It is shown that light can be perfectly confined in the photonic...     

Cancer Detection with Surface Plasmon Resonance-Based Photonic Crystal Fiber Biosensor

Plasmonics - In this study, early cancer detection of a single living cell is investigated by employing a surface plasmon resonance (SPR)-based photonic crystal fiber (PCF) biosensor structure. The...     

Bound States in the Continuum in a T-Shape Nanohole Array Perforated in a Photonic Crystal Slab

Plasmonics - We have studied the reflectance spectra of T-shape nanohole array in a layer of Photonic crystal (PhC) slab. Results show that light can be confined perfectly in the PhC slab at a...     

Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.     

Oligonucleotide Array for Identification and Detection of Pythium Species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

甲基毒死蜱对红细胞膜乙酰胆碱酯酶的抑制作用及其与膜脂相互关系

以人红细胞膜为材料,研究了甲基毒死蜱与膜上乙酰胆碱酯酶(AChE)的相互作用及其与膜脂的关系。结果显示,甲基毒死蜱对人红细胞膜AChE有明显的抑制作用,与膜温育30min,其半数抑制浓度约为0.10 mmol/L。动力学分析表明,其抑制作用为非竞争性。0.2％Triton X-100并不改变AChE对甲基毒死蜱的敏感性,亦即AChE上甲基毒死蜱的作用部位与其所处的脂质微环境无关。     

Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO₃ solvent (10⁻⁷ M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm⁻¹ was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g⁻¹ range (R² = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g⁻¹, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.     

基于悬液芯片系统的新城疫病毒强、弱毒高通量检测方法的建立

目的:利用悬液芯片系统建立一种高通量检测新城疫病毒强、弱毒的方法并将该方法的灵敏度与传统的酶联免疫反应(ELISA)进行比较.方法:将F48E9和LaSota单克隆抗体通过共价偶联的方式连接到聚苯乙烯微球的表面构成捕获抗体,利用捕获抗体、检测物、生物素化的多抗及链霉亲和素化的藻红蛋白建立双抗夹心的免疫检测模式.检测物作为抗原与捕获抗体结合后与生物素化的新城疫多抗进行反应,反应完成后,用链霉亲和素标记的荧光探针对反应产物进行标记得到悬液芯片系统的检测物.结果:微球包被实验结果表明,包被100 μL微球所需F48E9和LaSota单克隆抗体的最佳量分别是14.85 μg和17.65 μg;新城疫病毒多抗的最佳稀释倍数为400倍;悬液芯片检测方法检测NDV强毒的灵敏度为1∶160,弱毒的灵敏度为1∶320;抗体特异性实验表明,该方法所使用的两种捕获抗体的体异性良好.该方法与传统的ELISA在相同灵敏度的前提下,其在检测时间、检测步骤及高通量方面优于ELISA.结论:基于悬液芯片系统的新城疫强、弱毒高通量检测方法的建立对于该病毒的快速诊断具有重要的意义.     

Design of a Biosensor for the Detection of Dengue Virus Using 1D Photonic Crystals

Plasmonics - A biosensor for the detection of dengue virus has been designed using 1D photonic crystal. In the proposed structure [(Si/LiF)6D(LiF/Si)6], D is the defect layer. Refractive index of...     

Simultaneous Neutralization and Innate Immune Detection of a Replicating Virus by TRIM21

Tripartite motif-containing 21 (TRIM21) is a cytosolic immunoglobulin receptor that mediates antibody-dependent intracellular neutralization (ADIN). Here we show that TRIM21 potently inhibits the spreading infection of a replicating cytopathic virus and activates innate immunity. We used a quantitative PCR (qPCR)-based assay to measure in vitro replication of mouse adenovirus type 1 (MAV-1), a virus that causes dose-dependent hemorrhagic encephalitis in mice. Using this assay, we show that genetic ablation of TRIM21 or chemical inhibition of either the AAA ATPase p97/valosin-containing protein (VCP) or the proteasome results in a >1,000-fold increase in the relative level of infection in the presence of immune serum. Moreover, the TRIM21-mediated ability of antisera to block replication was a consistent feature of the humoral immune response in immunized mice. In the presence of immune sera and upon infection, TRIM21 also activates a proinflammatory response, resulting in secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These results demonstrate that TRIM21 provides a potent block to spreading infection and induces an antiviral state.     

液相芯片MASA技术用于儿童呼吸道感染病原学研究

利用多靶点液相芯片(Multi-Analyte Suspension Array,MASA)技术对引起儿童呼吸道感染(respiratory tract infections,RTI)的病原,包括人类呼吸道合胞病毒A型和B型(Human respiratory syncytial virus A and B, RSVA、RSVB)、严重急性呼吸综合征冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-CoV)、流行 性感冒病毒A型和B型(Influenza A virus and Influenza B virus,INFa,INFb)、副流感病毒1型和3型(Parain- fluenza virus 1 and 3,PIV1,PIV3)、衣原体(C．pneumoniae,CPN)和支原体(M．pneumoniae,MPN)进行了病原学 研究。我们采集并分析了140例患典型呼吸道感染症状儿童的咽拭子,发现在这些标本中至少被前述的一种病 原感染的标本有95例,阳性率为67．86％。结果显示这些标本中上述病原感染的情况分别为:RSVB感染的患儿 占35．71％、PIV3感染的占4．29％、INFa感染的占28．57％、INFb感染的占2．14％、MPN感染的占3．57％、CPN 感染的占17．86％,被两种以上病原混合感染的患儿有17．14％。这些标本中都没有检测到RSVA、PIV1和SARS- CoV病原的感染。RSVB病原的感染率在3岁以下的患儿中明显高于3岁以上的患儿,而INFa的感染情况则相 反;在上呼吸道感染患儿中检测到INFa病原感染的比例明显高于下呼吸道感染,而RSVB病原感染的情况相反。 此外,我们发现在2005年3月-5月中造成武汉地区儿童呼吸道感染的主要病原是以RSVB、INFa、CPN为主,而 RSVB感染则又是引起儿童下呼吸道感染和引起低龄儿童呼吸道感染的重要病原。     

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient’s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.