数字PCR技术的发展及应用

数字PCR(Digital PCR, dPCR)是继实时荧光定量PCR(Real-time quantitative PCR, qPCR)之后发展的高灵敏核酸绝对定量分析技术,通过把反应体系均分到大量独立的微反应单元中进行PCR扩增,并根据泊松分布和阳性比例来计算核酸拷贝数实现定量分析。与传统PCR技术相比,数字PCR 技术不依赖于标准曲线,具有更高灵敏度、准确度及高耐受性,可实现对样品的绝对定量分析。近年来,随着微流控技术日臻成熟,基于微流控技术的数字PCR技术得到了快速的发展,在基因突变检测、拷贝数变异检测、病毒微生物检测、转基因食品检测以及测序等方面均得到广泛的应用。本文对数字PCR的原理、技术发展和应用进行了概述。     

液滴微流控系统在数字聚合酶链式反应中的应用研究进展

数字聚合酶链式反应( PCR)技术近年来发展迅速。与以实时荧光定量PCR为代表的传统PCR技术相比,数字PCR技术显著提高了定量分析的精确度和灵敏度。数字PCR的快速发展与近年来微流控技术在数字PCR技术中的广泛应用有着密切的联系。早期的研究和商业化产品使用的是大规模集成流路微流控芯片,加工过程复杂且价格高昂。近年来,液滴微流控芯片被应用到数字PCR技术中,它可以在短时间内产生102~107个微液滴,每一个微液滴都是最多只含有一个目的基因片段的PCR反应器。 PCR扩增后,通过对单个微液滴的观察计数,就可以获得绝对定量的分析数据。本文综述了不同种类的液滴微流控系统在数字PCR技术中的应用,以及液滴数字PCR微流控芯片在生物、医药、环境等领域的应用。     

数字PCR在食品安全检测中的应用研究进展

聚合酶链反应(PCR)在动植物源掺杂鉴别、转基因成分和致病微生物等食品安全检测领域成为日趋重要的检测技术。从传统PCR、荧光PCR到数字PCR,PCR技术逐渐从定性分析、半定量分析发展到准确定量,不仅提升了准确度和检测效率,同时也扩展了食品检测范围,使食品安全的监管更加精细化。该文总结了近5年来数字PCR在食品安全检测中的研究进展,比较了传统PCR、荧光定量PCR和数字PCR的相关标准制订情况,列举、讨论了数字PCR在不同食品安全领域检测中的技术进展和存在的问题,并对数字PCR未来发展方向进行了展望。     

数字聚合酶链式反应技术及其在疾病检测中的研究进展

数字聚合酶链式反应（Digital polymerase chain reaction, d PCR）技术是通过将反应体系均分到数万个独立的PCR反应单元进行单分子级扩增,并结合泊松分布实现单拷贝核酸分子精确定量分析的PCR技术。dPCR技术具有灵敏度高、无需标准曲线等优势,被广泛应用于疾病检测领域。随着阶梯乳化和三维成像等技术的引入,dPCR技术在检测精度、多重检测和检测速度等方面得到极大改进,显著提升了其临床疾病诊断性能。本文追溯了dPCR技术的发展历程,对其在肿瘤和感染等疾病检测中的应用进行了综述,分析了dPCR技术发展存在的挑战与机遇,以期为dPCR技术未来的开发利用提供参考,推动临床检验分子技术高质量发展。     

数字PCR测定DNA含量及测量结果不确定度评定

数字PCR是一项不依赖校准物的DNA绝对定量技术,将含有DNA模板的反应溶液分配到大量独立的微室中并且进行扩增反应,通过统计反应室中的阳性信号来定量DNA的拷贝数。采用数字PCR技术对噬菌体λDNA的含量进行测定,并对测定结果进行不确定度评定。结果显示,由数字PCR测得的λDNA的含量为(2.43±0.35)μg/管(k=2),该结果与同位素稀释质谱法测量结果相吻合。     

聚合酶键式反应生物芯片/微装置的实时定量荧光检测

以微电子机械系统技术而发展起来的聚合酶链式反应(Polymerase Chain Re-action,PCR)生物芯片/微装置,由于具有所需样品和反应混合物体积少,反应速度快以及集成化程度高等优点而日益引起人们的重视。实时定量PCR是利用能特异标记PCR产物的荧光染料来动态显示PCR产物的累积,从而得到PCR扩增曲线的技术。本文介绍了实时定量检测技术及其在PCR生物芯片/微装置中的应用。     

Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

基于非接触电导检测的PCR液滴检测芯片的研究

数字PCR是继实时定量PCR之后新兴发展起来的一种绝对定量分析技术,广泛应用于疾病早期检测诊断和微生物检测分析等领域。然而,在数字PCR的微反应器检测环节,常规的光学检测方法需要复杂的光学设备,应用环境有限,成本高。因此研究了一种基于非接触电导检测的PCR液滴检测芯片,用电导检测取代传统的光学检测,有效降低了数字PCR结果读出系统的成本。通过优化电极结构与排布,采用三电极平行3D电极结构,减小了相对误差,提高了灵敏度,实现了无标记的PCR反应液滴阴性阳性比例检测。在DNA模板浓度为0.5×10~4～3×10~4 copies/μL内实现了良好的相关性(r~2=0.998),检测通量可达100个/秒。     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

Synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives

Different approaches for the synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives from simple amino acids have been investigated. A library of 33 precursors for the preparation of N-hydroxy pyrazinones was obtained in moderate to good yields.     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

A general and convenient synthesis of 4-(tosylmethyl)semicarbazones and their use in amidoalkylation of hydrogen,heteroatom, and carbon nucleophiles

A general synthesis of previously unknown semicarbazone-based α-amidoalkylating reagents, 4-(tosylmethyl)semicarbazones, has been developed. The synthesis involved three-component condensation of semicarbazones of aliphatic or aromatic aldehydes with the same or other aldehydes and p-toluenesulfinic acid. The scope and limitations of this reaction were investigated. The compounds obtained were demonstrated to be an efficient α-(4-semicarbazono)alkylating agents. They were reacted with H- (sodium borohydride), O- (sodium methylate), S- (sodium phenylthiolate), N- (pyrrolidine, sodium succinimide), P- (trialkyl phosphites), and C-nucleophiles (sodium diethyl malonate) to give the corresponding products of the tosyl group substitution, 4-substituted semicarbazones, including analogues of nitrofurazone. Among the prepared compounds tested in vitro for antibacterial and antifungal activity, three nitrofuryl-containing semicarbazones exhibited high biological activities with minimum inhibitory concentration (MIC) values of 8–32 μg/mL.     

Toward new camptothecins. Part 6: Synthesis of crucial ketones and their use in Friedländer reaction

In the context of the preparation of camptothecin and luotonin A analogs, the synthesis of some key keto-precursors and their use in Friedländer condensation are described. This paper also focuses on the stability of these keto intermediates and emphasizes the major differences between indolizinones and pyrroloquinazolinones series. Noteworthy is also the report of some original structures isolated as by-products of some experiments.     

Synthesis of novel chiral bidentate hydroxyalkyl-N-heterocyclic carbene ligands and their application in palladium-catalyzed Mizoroki–Heck couplings and asymmetric addition of diethylzinc to benzaldehyde

A small library of new chiral bidentate hydroxyalkyl-imidazolium salts 1 is conveniently synthesized on multi-gram scale from inexpensive and commercially available chiral pool amino acids. The corresponding carbenes, generated by deprotonation of imidazolium salts 1, in combination with palladium(II) chloride were tested in the Mizoroki–Heck coupling reaction. The most significant results in terms of yields and reactivities were achieved with low catalyst loading. The catalytic activities of these imidazolium salts were also investigated in the asymmetric addition of diethylzinc to benzaldehyde. The use of MgO nanoparticles as an additive in conjunction with these ligands played a crucial role in increasing the efficiency of these reactions.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.