免疫胶体金法提取环境标本中细菌DNA技术

将抗-DNA单克隆抗体标记在胶体金颗粒上制成免疫胶体金试剂,提取标本中DNA,直接用于PCR检测,从而建立一种简单、快速、高效的免疫胶体金方法提取环境标本中的DNA。结果表明：应用免疫胶体金试剂可有效去除环境标本中PCR抑制剂,浓缩模板,提高PCR检测敏感度3～4个数量级。操作步骤简单,无需使用有机溶剂,避免环境污染,吸附了DNA的免疫胶体金可直接用于PCR扩增。研制了免疫胶体金试剂并确定其最佳反应条件,有效提高PCR技术在检测现场环境标本中的敏感性和实用性。     

免疫胶体金法提取环境标本中细菌DNA技术*

将抗DNA单克隆抗体标记在胶体金颗粒上制成免疫胶体金试剂,提取标本中DNA,直接用于PCR检测,从而建立一种简单、快速、高效的免疫胶体金方法提取环境标本中的DNA。结果表明:应用免疫胶体金试剂可有效去除环境标本中PCR抑制剂,浓缩模板,提高PCR检测敏感度3～4个数量级。操作步骤简单,无需使用有机溶剂,避免环境污染,吸附了DNA的免疫胶体金可直接用于PCR扩增。研制了免疫胶体金试剂并确定其最佳反应条件,有效提高PCR技术在检测现场环境标本中的敏感性和实用性。     

一种快速提取植物病毒DNA的方法

介绍在PCR检测体系中一种快速提取病毒DNA的方法。利用高盐缓冲液溶液释放病毒DNA,同时利用葡聚糖凝胶微柱纯化提取液,有效消除样品中PCR抑制物质并直接作为PCR反应模板扩增检测病毒。该法无需任何特殊设备,适合对大量植株进行大通量的检测分析。     

植物叶片基因组DNA快速提取方法

为了满足分子生物学研究中大量植株基因需要PCR检测的需求,建立了一种无需研磨、无需有机试剂抽提的快速提取植物叶片基因组DNA的方法。通过比较基因组DNA的浓度及PCR检测结果,获得了最佳提取条件,即约50 mg叶片加入150μL含1%β-巯基乙醇的TE提取液,快速破碎1 min。破碎后的样品4℃13 500×g离心1 min,上清于-20℃冷冻后室温融解,4℃、13 500×g离心1 min,离心后收集的上清溶液即可用于PCR检测。整个提取过程仅需10-12 min,具有样品用量小、操作简单、廉价、高效等优点。使用此方法提取的烟草、水稻、大豆、玉米、油菜和花生的基因组DNA均可用于PCR扩增,并可成功扩增长度为3 244 bp的基因片段。此方法提取的基因组DNA也可用于对未知基因的扩增,获得4条新的花生actin基因序列。     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

实验动物皮肤病原真菌DNA快速少量提取法

PCR技术应用于实验动物皮肤病原真菌检测,方法简单、省时。但是,真菌的DNA提取较为困难。本文推荐一种既简单又经济快速的提取皮肤真菌DNA的方法,并能成功用于实验动物皮肤病原真菌质量检测研究。     

植原体DNA提取方法的改良

在总结多种植原体DNA提取方法的基础上 ,发展了一种提取植原体DNA新方法。用此方法提取的DNA经琼脂糖凝胶电泳检测到大于 15kb的DNA主带 ,基本无DNA碎带 ,不用RNase处理 ,也无RNA干扰 ,OD2 60 / 2 80 值显示产物纯度较高 ,无需任何处理 ,即可以作为模板扩增     

超声波法提取胎菊中绿原酸的研究

建立了胎菊中绿原酸的提取方法及检测方法.采用超声波法对胎菊中的绿原酸进行提取,考查了提取溶剂、提取时间、提取料液比对提取量的影响,HPLC测定提取液中绿原酸的含量,确定最佳提取工艺.结果表明,以甲醇为溶剂、提取时间为80 min、提取料液比为1:20时,胎菊中绿原酸的提取量最高.该方法简单、快速、准确,可用于胎菊中绿原酸的含量检测.     

以分子信标为报告分子的凝血酶检测新方法

以分子信标为报告分子,核酸适体为识别分子,发展了一种新的凝血酶检测方法.含有分子信标互补序列的核酸适体探针与凝血酶结合后,分子信标的荧光信号下降,从而得到凝血酶的浓度信息.该方法快速、灵敏,核酸适体探针无需荧光标记、设计简单,检测限达到0.83nmol/L.     

直接免疫荧光法检测狂犬病毒滴度的可行性研究

实验研究中建立用于定量检测狂犬病毒滴度的直接免疫荧光法,并将检测结果与传统小鼠脑内滴定法进行了比较,对照两种试验方法的灵敏度、特异性和重复性。结果显示,两种检测方法特异性基本一致;相同样品经多次检测的病毒滴度相近,重复性好、灵敏度高。直接免疫荧光法具有特异、灵敏、快速、操作简单、无需使用动物等优点,可应用于狂犬病毒滴度的定量检测。     

利用表面等离子共振传感器检测苏云金芽孢杆菌cry1F蛋白

目的：建立检测苏云金芽孢杆菌（Bt）crylF蛋白的表面等离子共振（SPR）传感器方法。方法：采用SPR检测技术,利用生物分子相互作用分析原理,在金表面修饰特异性单克隆抗体,对crylF蛋白的检测进行研究。结果：该方法可以较好地检测到crylF蛋白,最低检测限可达10ng／mL,并且具有很好的特异性。结论：SPR检测方法的重复性较好,灵敏度高,目前可用于crylF蛋白的定性检测,为crylF蛋白及其他Bt蛋白的检测提供了新方法,在检测转Bt基因植物方面具有广阔的应用前景。     

植物病毒检测技术──组织印迹法

组织印迹法（Tissue blotting）是在酶联免疫吸附（Enzyme-Linked immunosorbent Assay ELISA）的基础上发展起来的植物病毒检测技术,该技术不仅保持了ＥＬＩＳＡ对病毒检测的灵敏度高,特异性强的特点,而且大大地简化了操作程序,对病毒的检测更加快速、简单、方便、印迹在硝酸纤维素膜上的样品能保存３个月以上,检测结果能直观地显示出病毒感染的部位。组织印迹技术尤其适用于植物病毒的大规模普查。     

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

大肠杆菌变种的两种分子生物学检测方法分析

目的:对大肠杆菌的一种重要的变种--肠出血性大肠杆菌O157-H7的几种检测方法进行比较研究.方法:以自动免疫磁珠收集系统(AIMS)、自动酶标免疫测试系统(VIDAS)与传统常规的分离方法进行对比分析.结果:运用自动免疫磁珠收集系统(AIMS)方法对80份可能含有肠出血性大肠杆菌O157-H7的实样进行检测,检出份数为6份,检出率为7.5％,而且在一周之内可以全部对上述检出实样进行鉴定.AIMS法能够检出浓度在10cfu/mL模拟实样之中的肠出血性大肠杆菌O157-H7,然后将此法与CHROMagar 0157琼脂板相结合,其效果则更为明显.而自动酶标免疫测试系统(VIDAS)与传统与的分离方法则检测的效果不佳,检出率为0.自动免疫磁珠收集系统(AIMS)检测方法与自动酶标免疫测试系统(VIDAS)、传统与的分离方法在检出率方面存在显著的统计学差异,P＜0.01.结论:运用自动免疫磁珠收集系统(AIMS)结合CHROMagar 0157琼脂板对出血性大肠杆菌O157-H7进行检测,检出率较高、灵敏度较高且快速便捷,可以用于O157-H7外环境检测与食品污染源的实际调查之中,应该对其加以广泛地推广并应用.     

A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.     

RNA-templated DNA ligation for transcript analysis

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.     

Combination of aptamer with gold nanoparticles for electrochemical signal amplification: application to sensitive detection of platelet-derived growth factor

In this paper, we report a novel electrochemical detection approach for platelet-derived growth factor (PDGF) via "sandwich" structure and gold nanoparticles (Au-NPs) mediated amplification technique. The "sandwich" structure is fabricated based on the fact that PDGF has two aptamer-binding sites, which makes it possible for one PDGF molecule to connect with two aptamers simultaneously. It is found that this electrochemical system with "sandwich" structure and Au-NPs can significantly amplify the signal of electrochemical probe [Ru(NH(3))(5)Cl](2+) for PDGF detection, and thus increase the detection sensitivity significantly. As a result, this PDGF detection approach obtains an extraordinarily low detection limit of 1 x 10(-14)M for purified samples, 1 x 10(-12)M for contaminated-ridden samples or undiluted blood serum. This detection approach can also exhibit good stability and excellent specificity.     

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.     

Autoantibody biomarkers in the detection of cancer

By definition, tumor biomarkers are selective molecules that can distinguish between patients with cancer and controls. Serum tumor markers have been the most widely used approach for cancer detection. However, the limitations of these markers, which are based on the measurement of tumor antigens, preclude their general use in cancer screening and diagnosis. Here we give an overview of recent cancer biomarker developments based on the detection of autoantibodies produced against tumor antigens in patients' sera. This new detection method can measure the autoantibodies for a spectrum of tumor antigens in a single assay, with sensitivity and specificity exceeding those obtained using the conventional antigen determination method. Autoantibodies against serum cancer biomarkers offer a novel technology for cancer detection.