微流控芯片与基因诊断关系的研究进展

<正>微流控芯片(MC)也称为芯片实验室,其已经广泛于医学、生物、电子、流体、化学等领域~([1,2]),且MC可把样品制备、反应、分离、检测、扩增、分析等集成到一块几微米至几百微米尺度的芯片上并自动完成所有基本过程。目前,MC已经广泛地应用到医学基因诊断(GD)方面,例如基因多态性检测~([3])、基因高效性测序~([4])、基因快速性扩增~([5,6])等,为此,本文主要对MC与GD关系进行综述。1 MC简介1.1 MC相关概念MC主要是指在几微米至     

微流控芯片技术在心血管疾病研究中的应用

心血管疾病是威胁人类健康的重要疾病,具有很高的致死率和致残率,已成为现代医学研究的热点话题。将传统实验技术用于心血管疾病发生机制、诊断及治疗等方面的研究具有一定局限性。微流控芯片技术是一项新型实验研究手段,具有微型化、集成化、高通量、低消耗、高灵敏、分析速度快等特点,其在体外生物模型构建、生物分子检测、药物疗效评价及筛选等方面较传统技术具有优势,现已逐渐应用于医学生物学研究的多个领域。本文谨对微流控芯片技术在心血管疾病研究领域的应用进行简要综述。     

PCR-微流芯片法检测HBV基因型及其临床应用

根据HBV全基因序列异质性≥8％或S基因序列异质性≥4％，可将HBV分为A～H8种基因型。文献报道基因型存在一定的地理区域分布，并可能与感染途径、感染谱、疾病的进展有一定的相关性。我们采用PCR-微流芯片法对96例乙型肝炎患者进行HBV基因型检测，并与病毒指标、肝纤维化、肝功能等指标进行相关性分析，报道如下。     

基于微流控核酸等温扩增的登革病毒现场快速检测技术研究

[摘要]?目的?结合环介导等温扩增技术（loop-mediated isothermal amplification, LAMP）和微流控芯片技术,建立一种适合现场快速检测登革病毒的方法。方法?利用RT-LAMP,针对登革病毒基因组中3’非编码区中特异性序列进行扩增,建立基于微流控芯片技术的LAMP检测方法,优化检测体系,并对该方法的灵敏性和特异性进行评估。结果?基于LAMP 和微流控芯片技术的登革病毒检测方法,通过对病毒模板进行扩增,发现与其他病毒无交叉反应,特异性良好;同时,结果显示该方法对登革病毒检测灵敏性可达61.2 pg/μl,与实时荧光定量PCR仪所达到的检测灵敏性一致。结论?基于LAMP和微流控芯片技术的登革病毒检测方法具有操作简单、快速、对设备要求低等优势,并且灵敏性、特异性均较好,是一种便于开展现场快速检测的方法。     

生物芯片在病原体检测中的研究进展

生物芯片作为一种高通量的基因检测技术,在多个研究领域已广泛应用,本文介绍了生物芯片技术的基本原理,综述了其在病原体检测中的研究进展。     

环介导恒温扩增芯片法在下呼吸道感染病原体检测中的临床应用

目的分析环介导恒温扩增芯片法(LAMP)在下呼吸道感染病原体检测中的应用价值。 方法选择2018年1月至2018年9月空军军医大学第二附属医院收治的1 092例疑似下呼吸道感染患者,分析支气管肺泡灌洗液病原体检测结果,以细菌培养结果为金标准,评价LAMP检测8种常见下呼吸道感染病原体的灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比、阴性似然比。 结果LAMP检测8种病原体的灵敏度差异较大,检测金黄色葡萄球菌灵敏度最高(100%),检测大肠埃希菌灵敏度最低(40%),检测8种病原体的特异度均高于94%。LAMP检测8种病原体的阳性预测值低于65%,阴性预测值高于98%,阳性似然比高于13,阴性似然比各病原体间差异较大,金黄色葡萄球菌阴性似然比最低(0.00),大肠埃希菌阴性似然比最高(0.60)。 结论LAMP在下呼吸道感染病原体检测中有较高的准确性,利于下呼吸道感染的早期诊断和精准治疗,具有临床意义。     

应用多聚酶链反应检测腹泻病原体

腹泻为婴幼儿常见病。明确腹泻病原体可为诊断、治疗和预防提供可靠的依据,因而研究快速、简便,敏感而准确的检测腹泻病原体的方法具有重要意义。 传统检测腹泻病原体方法是体外培养,费时数天乃至数周,还有些病原体不易体外培养。免疫学方法由于宿主半抗原与相关病原体的交叉反应,给诊断带来困难。近年来基因探针检验技术有很大发展,但该     

冠状病毒检测新方法及其在其他病原体检测中的应用前景

冠状病毒与人和动物的许多疾病有关。迄今至少已发现了13个种属多个抗原表型的冠状病毒，其中人冠状病毒包括HCoV OC43和HCoV 229E两个抗原型，是引起人类上呼吸道感染的重要病原体。2003年严重急性呼吸系统综合症(Severe Acute Respiratory Syndrome，SARS)在中国和世界上其他国家爆发流行，其病原体SARS相关冠状病毒(SARS-CoV)作为新的病毒亚种引起了社会的广泛关注。     

微流芯片检测HBV前C区nt1896/BCP区nt1762基因突变的临床意义

目的探讨HBV前C区nt1896/BCPnt1762基因突变的临床意义。方法在418例血清HBeAg阴性/抗-HBe阳性的慢性乙型肝炎患者,采用微流基因芯片检测HBV前C区nt1896/BCPnt1762基因突变。同时观察血清ALT患者、HBV DNA水平和临床表现。结果nt1896变异195例(46.7%),1762变异192例(45.9%),ALT>正常上限值198例(47.4%),HBV DNA>1×105copiel/ml150例(35.8%),乏力,纳差,肝区胀痛213例(50.9%)。结论HBV前C区nt1896/BCP1762基因突变与HBeAg阴性患者ALT异常,HBV DNA升高及其临床表现具有相关性。     

问号钩端螺旋体检测基因芯片的研制

目的　制备一种用于快速检测、鉴定问号钩端螺旋体的DNA微阵列芯片。方法　从Genbank中选取钩体 2 3SrDNA序列 ,设计一对种特异性引物和DNA探针 ,通过Blast的基因同源性比较 ,验证引物和探针的通用性与特异性。将合成的探针点样到玻片上制备成基因芯片。用Cy3标记引物 ,通过不对称PCR反应获取荧光素标记的靶序列 ,然后与制备的芯片杂交。结果　设计的引物与探针仅同时与问号钩体 2 3srDNA完全同源。该引物可扩增我国 18个血清群的 2 5株钩体 ,均出现单一 4 82bp的扩增产物 ,而双曲钩体及其他螺旋体、病原、空白对照均无任何DNA扩增条带。芯片杂交结果与常规PCR方法结果一致 ,敏感性高于PCR。结论　基因芯片可以快速、灵敏、特异地检测问号钩端螺旋体。     

脱嘌呤/脱嘧啶核酸内切酶1在肝细胞癌中的表达及其意义

肝癌(HCC)是我国最常见的恶性肿瘤之一,其癌变是一个多步骤的渐进过程.脱嘌呤/脱嘧啶核酸内切酶(APE)1,可修复损伤的DNA.APE1又名氧化还原因子1(Ref-1),可调节许多转录因子的DNA结合活性.我们采用免疫组织化学及原位杂交方法检测APE1在HCC中的表达情况,旨在探讨APE1基因表达在HCC的发生及恶性转化中的作用与地位.     

Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages

Many antagonistic interactions between hosts and their parasites result in coevolution. Although coevolution can drive diversity and specificity within species, it is not known whether coevolutionary dynamics differ among functionally similar species. We present evidence of coevolution within simple communities of Pseudomonas aeruginosa PAO1 and a panel of bacteriophages. Pathogen identity affected coevolutionary dynamics. For five of six phages tested, time-shift assays revealed temporal peaks in bacterial resistance and phage infectivity, consistent with frequency-dependent selection (Red Queen dynamics). Two of the six phages also imposed additional directional selection, resulting in strongly increased resistance ranges over the entire length of the experiment (ca. 60 generations). Cross-resistance to these two phages was very high, independent of the coevolutionary history of the bacteria. We suggest that coevolutionary dynamics are associated with the nature of the receptor used by the phage for infection. Our results shed light on the coevolutionary process in simple communities and have practical application in the control of bacterial pathogens through the evolutionary training of phages, increasing their virulence and efficacy as therapeutics or disinfectants.Many host–parasite associations coevolve, and patterns in this antagonistic interaction are influenced by biology and environment (1–3). In single-species host–parasite interactions, parasite genotypes show differences in their host ranges and specificities on host genotypes, providing the basis for such coevolution (4–8). Arms race dynamics (ARD) driven by directional selection favors a broader resistance range in the host against a greater number of parasite genotypes and an increased host range in the parasite allowing more host genotypes to be infected (2, 9). In contrast, fluctuating selection dynamics (FSD), in which there is no directional change in the evolution of the host resistance range, is governed by negative frequency-dependent selection, favoring hosts that resist the most frequently encountered parasite genotypes and parasites that infect the most common host genotypes (9–11). It has been suggested that ARD predominates during the initial stages of coevolution, when adaptations to the coevolving opponent are largely cost-free, whereas FDS is more significant at later stages, when attack/defense alleles accumulate in the genome and impose costs (12). Thus, when systems shift from ARD to FSD, dynamic coevolutionary equilibria may arise, with constant numbers of attack/defense alleles at the individual level (13) and the continuous frequency-dependent (re)cycling of alleles at the population level (14).Coevolutionary dynamics between hosts and parasites is increasingly investigated using experimental evolution (15) and more specifically time-shift assays (16–18), in which hosts or parasites from a given time point are compared with their counterparts from the past or future, enabling the examination of putative reciprocal adaptations (19, 20). Some of the prime experimental models are bacteria and their lytic phages, which exhibit rapid evolution and are amenable to time-shift tests. Recent study indicates that coevolving populations may exhibit either ARD or FSD (12, 21–24), and there is some evidence for the genetic mechanism involved [e.g., mutations at tail fiber genes (12, 25)] and for the expansion of the phage host range as coevolution proceeds (6). Most studies of bacteria–phage coevolution involve the model system Pseudomonas fluorescens SBW25 and its lytic phage ϕ2 (15). Although these studies are important for an in-depth understanding of this process, their restriction to a single host–parasite pair is unfortunate, given the immense diversity of bacteria and phages in both terrestrial and aquatic ecosystems (26–28) and the importance of many of these organisms in human health (29). Thus, the generality of previous results to other bacterial species and to different phages parasitizing a given bacterium remains an open question.Coevolution may be particularly important in an applied context, namely when predators or parasitic organisms are used for biocontrol (30). For instance, Conrad et al. (31) recently argued that a community perspective with its ecological and evolutionary underpinnings is needed to explore the usefulness of phages as antimicrobial agents to treat cystic fibrosis patients infected with several bacteria and notably strains of Pseudomonas aeruginosa, a congeneric to the model organism P. fluorescens. Different phages naturally have different host ranges (8, 32, 33), but whether phage taxonomic origin influences impacts on P. aeruginosa is not known. Despite the study of phage mixtures to control P. aeruginosa infections (34), the nature of bacterial cross-resistance to phages other than that with which the bacterium evolved has not been addressed.Previous studies are inconclusive regarding whether P. aeruginosa coevolves with bacteriophages (35, 36). Given the ubiquity of this model organism in natural habitats (37) and as a widespread pathogen in hospitals (38, 39), it is important to know whether phage parasitism influences P. aeruginosa population biology and adaptation, whether coevolution between these antagonists actually occurs, and, if so, whether there are general patterns shared by different phages. Here, we test coevolutionary dynamics and their consistency in a panel of lytic bacteriophages and their host P. aeruginosa PAO1. We allowed this bacterium to interact and potentially coevolve with each of six different phage isolates separately, four from the Podoviridae and two from the Myoviridae (Table S1), for a total of 10 serial transfers (∼60 generations). Using bacteria and phages isolated from different time points, we conducted time-shift assays of resistance to infer patterns of the coevolutionary process. Finally, to assess specificity, we performed a cross-resistance assay with evolved bacteria and the six ancestral phage isolates to compare resistance of the bacteria to their “own” phage and to “foreign” phages with which they had not coevolved.     

Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Rapid, specific, and sensitive measurements of target analytes are the goals of many methods used in molecular biology and biotechnology. Bulk methods typically use a binding molecule to recognize the target molecule, combined with indirect optical reporter mechanisms, such as fluorescent dye labels or changes in bulk optical properties resulting from target binding. Such classical methods detect an average over many molecular binding events, and over timescales much longer than that of the primary molecular interactions. In contrast, the binding interactions of interest fundamentally occur at the single-molecule level, and are typically dynamic and stochastic in time, and thus contain far more detail than what is reflected in bulk reaction rates. This highlights the potential to access a fundamentally richer and more powerful level of information when measuring molecular interactions.Approaches to observing the details of single-molecule interactions fall into categories based on the detection method (1). Many are fluorescence-based single-molecule optical biosensors (2–5), although other specialized physical techniques have been used, including electrochemical sensors (6, 7), plasmonic sensors (8), surface-enhanced Raman spectroscopy (9), and methods coupled to nanopore detection (3, 10, 11). In addition to complications of labeling procedures needed to add fluorescent reporters to targets of interest, single-molecule optical methods suffer from fundamental limitations in signal and resolution. A major challenge for single-molecule fluorescence methods is obtaining high signal-to-noise ratio, because the rate of photon production from single dye molecules is restricted by illumination intensity limits and photo-bleaching (12), constraining both short-time and long-time measurements. Molecular motion effects and diffraction also limit the ultimate spatial resolution or density of multiplex optical reporters.Moving away from photon-based detection to all-electronic detection can remove these fundamental constraints on signal-to-noise ratio, scaling, and bandwidth, and moreover is maximally compatible with implementation on modern semiconductor chip devices. It would be advantageous to measure molecular interactions on a complementary metal-oxide semiconductor (CMOS) chip to leverage their low-cost mass manufacturing, speed, and miniaturization. These are hallmarks of modern CMOS chip-based devices, such as portable computers and cell phones. Such on-chip devices also enjoy a durable roadmap for future improvements provided by 50 years of Moore’s Law scaling of CMOS chips, and corresponding chip foundry infrastructure and supply chains.However, the full potential of this vision of moving molecular biosensing “on-chip” can only be realized by using a suitably compatible sensor concept. To this end, there are three fundamental sensor design principles to consider: manufacturability, scalability, and universality (see SI Appendix). The field of molecular electronics provides a conceptual solution to all of these challenges, wherein a single-molecule in a circuit would provide the fully scaled sensor to solve the More-than-Moore scaling problem common to sensor devices. Scientific advances on the electrical properties of molecules, as well as bioelectronic inspirations, led to the proposal in the early 1970s that single molecules could be engineered for use as circuit elements (13), to perform circuit functions such as a rectifier or switch. Due to limitations of nanofabrication technology, it was not until the late 1990s that the first single-molecule circuits were demonstrated experimentally (14). Interest in this field of molecular electronics expanded dramatically after that point (15–18), and it was proclaimed the scientific breakthrough of the year by Science in 2000 (19). There it was noted that integrating molecules into chips would be the critical advance needed for this new field to have broad impact.The experimental study of single-molecule electronic sensing was initially based on carbon nanotube (CNT) sensor devices. Their potential as sensors for single-molecule interactions became apparent (20) initially in the context of sensing gas molecules (21) and then chemical reactions (22). Nuckolls, Shepard, and colleagues (23–25) introduced a single-molecule sensor for DNA–DNA binding (hybridization) processes, by functionalizing a CNT with a single DNA oligomer “probe” molecule. Collins, Weiss, and colleagues (26–28) showed that a CNT can be used for real-time monitoring of the activity of a single enzyme molecule attached to nanotube, including DNA polymerase enzymes. Unfortunately, at present there is no way to mass manufacture CNTs having precise structure and functionalizations, and despite decades of attention (20), there is also no established path to integrating them into manufacturable CMOS chip devices (29, 30). Thus, while CNT molecular wire sensors enabled pioneering work on single-molecule sensing, they do not satisfy the design principles for a CMOS chip sensor platform.In contrast, an ideal molecular wire should allow precision engineering to provide a site-specific conjugation moiety for attachment of probe molecules, as well as to provide suitable end groups for self-assembly into the nanoelectrodes on a CMOS chip, and should be readily available through existing manufacturing processes. This limits the candidates to peptides, proteins, or DNA as molecular wires, as these are in fact the only conducting polymers for which there are well-developed precision synthesis capabilities, including extensive means of precision functionalization. Double-stranded DNA (dsDNA) helices (31–35) and protein α-helices (36–45) have both been studied as molecular wires. Examples from direct current measurements through various (short) α-helixes in the literature (42) suggest a (long) 25-nm α-helix could exhibit currents in the broad range of 3 picoamp (pA) to 120 pA at 1 volt bias, depending on the amino acid sequence, buffer conditions, and the nature of the peptide–metal attachment. Detailed tunneling probe methods have also recently been used to study conduction through larger proteins (46–49). While not nearly as conductive as CNTs, these biopolymers have the great advantage for present purposes of allowing precision engineering using existing manufacturing capacity.     

膜芯片检测人B组、C组轮状病毒的初步研究

目的建立检测人B组、C组轮状病毒（RV）的膜芯片。方法采用化学合成法合成B组、C组RV高度保守的核酸扩增区域,以地高辛标记上游引物,经半巢式PCR扩增B组、C组RV的保守检测区域,采用本实验室自行研制的膜芯片进行杂交检测。结果B组、C组RV的扩增产物同膜芯片上特异探针呈高度特异杂交。结论自制的膜芯片检测技术能够实现B组、C组RV的检测．并具有特异性好、高通量、平行性和简便快速的特点。     

Health Technology Assessment of pathogen reduction technologies applied to plasma for clinical use

Although existing clinical evidence shows that the transfusion of blood components is becoming increasingly safe, the risk of transmission of known and unknown pathogens, new pathogens or re-emerging pathogens still persists. Pathogen reduction technologies may offer a new approach to increase blood safety. The study is the output of collaboration between the Italian National Blood Centre and the Post-Graduate School of Health Economics and Management, Catholic University of the Sacred Heart, Rome, Italy. A large, multidisciplinary team was created and divided into six groups, each of which addressed one or more HTA domains.Plasma treated with amotosalen + UV light, riboflavin + UV light, methylene blue or a solvent/detergent process was compared to fresh-frozen plasma with regards to current use, technical features, effectiveness, safety, economic and organisational impact, and ethical, social and legal implications. The available evidence is not sufficient to state which of the techniques compared is superior in terms of efficacy, safety and cost-effectiveness. Evidence on efficacy is only available for the solvent/detergent method, which proved to be non-inferior to untreated fresh-frozen plasma in the treatment of a wide range of congenital and acquired bleeding disorders. With regards to safety, the solvent/detergent technique apparently has the most favourable risk-benefit profile. Further research is needed to provide a comprehensive overview of the cost-effectiveness profile of the different pathogen-reduction techniques. The wide heterogeneity of results and the lack of comparative evidence are reasons why more comparative studies need to be performed.     

基因芯片技术及其在寄生虫学研究中的应用

随着人类基因组计划(human genome project,HGP)和一些模式生物基因组计划的完成,基因序列数据正以前所未有的速度迅速增长,对基因组学的研究已从结构基因组学逐步转向了功能基因组学.由于基因芯片技术操作简便,获得的信息高度特异、稳定,在寄生虫学研究领域已得到广泛应用.随着寄生虫分子遗传学研究的进展和寄生...