荧光偏振与非对称基因扩增技术联用分析全血XPD基因单核苷酸多态性

荧光偏振检测技术是一种新型的基因诊断技术,不需要分离结合和未结合的探针,不依赖荧光强度,具有反应速度快、效率高、准确及成本低等优点,尤其在检测小分子领域具有很强的优势.     

基于磁性颗粒微阵列与双色荧光杂交的单核苷酸多态性分型方法研究

基于磁性颗粒微阵列与双色荧光杂交,建立了单核苷酸多态性(Single nucleoitide polymorphism,SNP)分型方法。将利用不对称扩增得到的含有待检测位点生物素标记的单链PCR产物固定在链亲和素修饰的金磁纳米颗粒(Gold magnetic nanoparticles,GMNPs)表面;将ssDNA-GMNPs混合物点样在底部固定有磁铁的载玻片上构建磁性颗粒微阵列,然后在基因框中与双色荧光探针杂交;杂交完全后,充分洗涤,通过扫描获得分型结果。通过优化不对称PCR的扩增条件,直接扩增出产量较高的单链DNA作为靶序列用于分型。利用本方法对24个样本MTHFR基因的C677T位点多态性进行了检测。实验证明,本方法步骤简单,易实现自动化操作、非常适用于分子诊断与法医鉴定。     

实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性

建立了实时荧光聚合酶链式反应( PCR)偶联高特性核酸侵入反应检测单核苷酸多态性( SNP)的方法。优化了体系中flap核酸内切酶1(FEN1酶)和野生型检测探针等用量,确定了最佳反应条件,即FEN1酶用量为1.5 U,野生型检测探针用量为0.125μmol/L,0.5μmol/L Invader突变型检测探针,各0.25μmol/L通用野生型( VIC)和突变型( FAM)荧光共振转移发卡探针,显著降低了野生型样本和突变型样本背景信号,避免了背景信号对检测结果分型的干扰。采用本方法对编码乙醛脱氢酶2( ALDH2)基因ALDH2*2位点21例样本、细胞色素P4502C19基因CYP2C19*2和CYP2C19*3位点各19例样本进行分型检测,结果表明, AL-DH2*2位点GG纯合10例,GA杂合8例,AA纯合3例;CYP2C19*2位点GG纯合9例,GA杂合8例,AA纯合2例;CYP2C19*3位点GG纯合18例,GA杂合1例。使用焦磷酸测序进行验证,两种方法检测结果一致。本方法特异性好、操作简便、耗时短、成本低,可实现对SNP单管闭管无污染的分型检测。     

荧光偏振免疫分析技术的研究进展

对荧光偏振免疫分析技术的研究进展作了详细的评述。介绍了荧光偏振免疫分析技术的基本原理和研究热点,评述了它在临床检验、环境与食品监测和农药残留量分析等领域中的应用,对荧光偏振免疫分析方法的优缺点作了总结,引文献45篇。     

基于核酸脱碱基位点的铽离子荧光增强型单核苷酸多态性识别研究

基于核酸脱碱基(AP)位点构建了无机配体稀土铽离子(Tb^3＋)荧光增强型单核苷酸多态性(SNP)识别方法. 在目标链靶标碱基对应的探针链上相应位置引入AP位点, 发现Tb^3＋可以选择性地结合在AP位点, 光激发时发生从DNA碱基到结合的Tb^3＋的能量转移, 使Tb^3＋特征荧光显著增强. 这种荧光增强作用与靶标碱基及AP位点侧翼碱基类型密切相关. 当靶标碱基和侧翼碱基为G时, 荧光最强. 该方法可用于区分肿瘤抑制基因p53密码子177位的C/G碱基变异.     

一种同时检测多个单核苷酸多态性位点的新方法

对人类基因组中的单核苷酸多态性进行快速而准确的定位和分型是人类基因组计划的重要内容。本文利用SNaPshot试剂盒，建立了一种以多重引物延伸为基础、可同时对多个已知单核苷酸多态性位点进行快速而准确的遗传分型的方法。利用这一方法检测了20例大肠癌病人肿瘤组织中错配修复基因hMLHl和抑癌基因P53的8个单核苷酸多态性位点(包括5个突变热点)，其中一例发现错配修复基因hMLHl的384位密码子处发生GTT→GAT的杂合性突变。对该基因外显子的直接测序结果与SNaPshot检测结果完全吻合，充分证明了该方法的可靠性。     

人类基因组的单核苷酸多态性及其研究进展

对单核苷酸多态性(SNP)的概念及特性作了简要说明,介绍了14种检测SNP的方法,讨论了各种方法的原理及优缺点,并对目前SNP在遗传图的绘制、遗传病致病基因的搜寻、药物设计及法医学等方面的应用及其研究进展进行了综述。引用文献29篇。     

单核苷酸多态性电化学生物传感检测研究进展

作为第3代遗传标记物,单核苷酸多态性分析在实现遗传疾病的早期医学诊断以及发病机理研究等方面具有非常重要的意义,已成为当今分析化学和生命科学研究的热点领域。电化学生物传感器因具有灵敏、检测装置轻便价廉且易于微型化、自动化等优点,近年在单核苷酸多态性检测分析中得到了迅速的发展。该文简述了单核苷酸多态性生物传感检测的识别原理、电化学生物传感检测方法及信号放大策略,综述了近年来的研究进展,并对未来的发展方向作了展望。     

一种基于适配分子的偏振荧光检测法

凝血酶适配分子（aptamer)可与凝血酶以高亲和力结合，二者的结合可改变荧光素标记的适配分子溶液的荧光偏振强度，据此建立了适配分子荧光偏振法测定血酶的新方法。方法的线性范围是0.1-4IU/mL；检出限为0.09IU/mL。将该方法用于人体血浆中凝血酶含量分析，结果满意。     

Development of Effective Skin Cancer Treatment and Prevention in Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is a rare, recessively transmitted genetic disease characterized by increasingly marked dyspigmentation and xerosis (dryness) of sun‐exposed tissues, especially skin. Skin cancers characteristically develop in sun‐exposed sites at very much earlier ages than in the general population; these are often multiple and hundreds or even thousands may develop. Eight complementation groups have been identified. Seven groups, XP‐A…G, are associated with defective genes encoding proteins involved in the nucleotide excision DNA repair (NER) pathway that recognizes and excises mutagenic changes induced in DNA by sunlight; the eighth group, XP‐V, is associated with defective translesion synthesis (TLS) bypassing such alterations. The dyspigmentation, xerosis and eventually carcinogenesis in XP patients appear to be due to their cells’ failure to respond properly to these mutagenic DNA alterations, leading to mutations in skin cells. A subset of cases, especially those in some complementation groups, may develop neurological degeneration, which may be severe. However, in most XP patients, in the past the multiple skin cancers have led to death at an early age due to either metastases or sepsis. Using either topical 5‐fluorouracil or imiquimod, we have developed a protocol that effectively prevents most skin cancer development in XP patients.     

高效液相色谱-荧光检测法同时测定全血中5种香豆素类杀鼠剂

建立了同时测定全血中杀鼠灵、杀鼠迷、溴敌隆、氟鼠灵与溴鼠灵等5种香豆素类杀鼠剂的简便、灵敏、准确的高效液相色谱-变波长荧光分析方法。全血样品经乙酸乙酯提取后,在XDB C18柱(150 mm×2.1 mm, 5 μm)上以甲醇-0.2%乙酸水溶液(体积比为88∶12)混合液为流动相,采用荧光变波长程序检测,同时通过建立各杀鼠剂的荧光光谱库,利用谱库检索,提高定性的准确度。各杀鼠剂的线性范围为0.01~10.00 mg/L（杀鼠灵为0.05~10.00 mg/L）,检出限为0.01~0.05 mg/L,方法的加标回收率为81%～98%,其相对标准偏差（RSD）为3.8%～8.5%。该法适用于中毒病人的中毒诊断检测。     

湿法消解-原子荧光法测定全血中硒

以全血样品为原料,探讨湿法消解-原子荧光法测定全血中的硒含量。血样经硝酸-高氯酸消解后,用硼氢化钠将硒还原成硒化氢,由氩气载入原子化器,产生的原子荧光强度与试液中硒元素含量在一定范围内呈正比,外标法定量。以消解效率为指标,优化样品的消解条件,测定血中硒在0-10μg/L范围内线性情况良好,相关系数为0.9992,最低检出限为0.143μg/L,相对标准偏差为1.51%-1.58%,平均加标回收率为90.86%-104.62%。血中硒的原子荧光测定法灵敏度高,精密度和稳定性好,可应用于血中硒的生物监测。     

基于荧光偏振的高灵敏度蛋白激酶活性分析

利用TiO₂纳米棒与磷酸化肽的特异性相互作用,基于荧光偏振检测,建立了快速、简便的高灵敏度蛋白激酶活性分析方法. 在蛋白激酶催化作用下,荧光标记的肽底物被磷酸化,磷酸化的荧光肽通过磷酸基团特异性结合在TiO₂纳米棒表面,从而使底物肽上标记的荧光分子的旋转速率发生改变,通过对荧光偏振度进行测量,可实现蛋白激酶活性的定量检测,该方法对蛋白激酶A(PKA)的检出限可达0.0004 U·μL^-1. 此外,该方法还成功用于PKA抑制剂H-89的检测,在基于蛋白激酶抑制剂的靶向药物筛选方面具有很好的应用前景.     

One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

Abstract

The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs was investigated. It was successfully shown that even one mismatch could be detected using fluorescence polarization analysis if the mismatch position was on the base that pairs with the probe DNA at the 5′ terminus where fluorescein isothiocyanate (FITC) is attached.     

基于核酸适配体的荧光法检测水胺硫磷和丙溴磷

建立了基于适配体的农药水胺硫磷和丙溴磷的荧光检测方法.采用可特异性识别水胺硫磷和丙溴磷、且5 '端标记荧光基团FAM的核酸适配体(F-ssDNA),与3 '末端标记猝灭基团DABCYL的短链序列(Q-ssDNA)互补杂交形成双链结构,荧光基团的荧光被淬灭,荧光信号很弱;此时加入靶分子,特异性结合核酸适配体,引起互补短链序列从双链结构中解离,使适配体荧光信号增强,基于此可实现水胺硫磷、丙溴磷的定量检测.优化后的检测条件为:将终浓度为25 nmol/L F-ssDNA与50 nmol/L Q-ssDNA在25℃孵育20 min,使二者杂交形成双链适配体探针复合物,加入等体积的农药样品孵育60 min,然后检测体系的荧光信号变化值△I.在最佳条件下,△I与水胺硫磷和丙溴磷的浓度均在50～ 500 μmol/L范围内呈线性关系.水胺硫磷的检出限(LOD,3σ)为11.4 μmol/L,相对标准偏差(RSD)为5.8％(n=10);丙溴磷的检出限为14.0 μmol/L,RSD为4.9％(n=l0).用于实际水样中两种农药的检测,加标回收率为85.8％ ～95.3％.     

A Fluorescence Polarization Assay of Erythrocyte Membrane Fluidity on the Abbott TDx Analyzer

Four fluorescein dye derivatives (attached to cholic acid, deoxycholic acid, lithocholic acid, and 5-cholenic acid-3β-ol) were studied as a means of detecting alterations in erythrocyte membrane fluidity. In addition, membrane fluidity using these novel dye derivatives was correlated to nonenzymatic glycation of erythrocyte membrane ghosts. These dyes have excitation and emission wavelengths suitable for analysis on an Abbott TDx analyzer. Both the lithocholic acid and the 5-cholenic acid-3β-ol fluorescein derivatives were capable of detecting erythrocyte membrane fluidity with an increase in polarization of approximately 34% over the range of glycation studied, and the fluidity was strongly correlated with the degree of glycation. The cholic acid and deoxycholic acid fluorescein derivatives were unable to enter the erythrocyte membrane since no alteration in fluorescence intensity or polarization was observed upon incubation with the membrane preparations. The results obtained indicate that the lithocholic acid and 5-cholenic acid-3β-oI fluorescein derivatives are potential new reagents for the development of routine assays for membrane fluidity on the Abbott TDx analyzer.     

Detection of Single Molecule on Water Surface by Confocal Fluorescence Microscopy

In the past decade,several research groups have succeeded in observing single molecule in liquid, and more recently at silica surface in the near-field mode as well as at polymer-air interface in far-field mode. Due to the significance of air-water interface in surface chemistry, we prospect that research works on single molecule detection (SMD) of the air-water surface could open a new era in surface photochemistry and photophysics.     

适体荧光分析法检测养殖废水中四环素类抗生素

建立了核酸适体识别-荧光探针技术检测养殖废水中土霉素、四环素、金霉素及强力霉素4种四环素类抗生素(TCs)总残留量的新方法。两段DNA对TCs共同识别后折叠成稳定的"发卡型"双链结构,核酸染料4'-6-二脒基-2-苯基吲哚(DAPI)能插入"发卡"部位产生荧光信号发射,据此可实现对TCs的定量检测。在最优实验条件下,以土霉素为目标分子建立工作曲线,在10~50 nmol/L范围内,荧光强度与对应浓度呈良好线性关系,土霉素的检出限为2.3 nmol/L。用四环素、土霉素、金霉素和强力霉素分别进行加标回收实验,平均回收率为88.5%~102.3%,相对标准偏差(n=5)为3.2%~6.7%。5个养殖场水样中均有TCs检出,检出量在7.6~42.7 nmol/L之间。该方法简单、灵敏、快速,可满足养殖场废水中TCs总残留量的测定要求。