四种结构类型的蛋白质设计方法

给出了以疏水一亲水模型为基础的蛋白质设计方法,该方法以物理学原理为基础,以相对熵作为优化的目标函数。对四种不同结构类型的天然结构的真实蛋白质进行了检测,分析了影响检测成功率的主要因素,结果表明,该方法是普适的,可用于对不同结构类型的蛋白质设计序列。     

病毒中和抗体检测方法研究进展

疫苗接种在病毒性传染病防控中发挥了重要的作用,有效的疫苗在机体内能够快速诱导高效价的中和抗体,中和抗体水平是衡量疫苗免疫保护效果的关键指标。为应对疫苗的快速研发、大规模临床试验检测以及疫苗接种后人群免疫原性检测的需求,急需建立高灵敏度、高通量的病毒中和抗体评价方法。目前病毒中和抗体检测方法除传统的以活病毒为基础的噬斑抑制法和细胞病变法外,结合新技术发展,已经有很多新的方法,如含有报告基因的重组活病毒的检测方法和以假病毒为基础的中和抗体检测方法等,现对病毒中和抗体检测方法的研究作一综述。     

沙门氏菌检测方法的研究进展

沙门氏菌作为常见的人兽共患病原菌之一,不仅会引起各种动物疾病,而且与人类多种疾病有关,其中,由沙门氏菌引起的食物中毒显得尤为突出。因此沙门氏菌的研究和探讨显得极为重要,而其检测方法则是研究的核心。为此,笔者查阅国内外文献,综述沙门氏菌检测方法,认为,沙门氏菌现行的主要检测方法包括三个方面:传统标准检测法、免疫学法和分子生物学法。传统方法鉴定沙门氏菌耗时较长;以抗体为基础免疫学方法将检测时间缩短了一半,且灵敏性高和特异性强;以核酸为基础的PCR技术由于灵敏、简单、快速和特异已被广泛用于沙门氏菌检测。     

基于发光共轭聚合物的核酸生物传感器

本文介绍了以发光共轭聚合物为基础,以荧光为检测手段,针对核酸进行特异性识别的一类检测信号放大的生物传感器。主要介绍了DNA和RNA的几种检测方法,探讨了影响荧光传感信号的因素。     

一种新的以影像为基础的细胞增殖和细胞毒性检测方法

3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑盐)（MTT）比色法是传统上检测细胞增殖和细胞毒性的常用方法.
CloneSelectTM成像系统是一种以影像为基础的用于分析细胞生长的可视检测系统.本研究采用人结直肠癌HCT116细胞系,运用CloneSelect成像系统和MTT方法分别检测药物阿的平的细胞毒性,并采用Bland Altman作图法比较两种实验方法获得的pEC50值,分析两种研究方法获得的结果的一致性. 结果表明,CloneSelectTM成像系统和MTT法获得的pEC50值具有较好的一致性.与MTT方法相比,基于影像的CloneSelectTM成像分析技术检测快速、无损伤且结果更准确,获取资料不损伤细胞,允许后续其它时间点或动力学检测. 研究提示,这种新的以影像为基础的检测技术可以替代MTT方法,用于分析不同药物的抗细胞增殖活性.     

《柳叶刀-肿瘤学》：高危HPV DNA检测可初筛宫颈癌

高危型人乳头瘤病毒（HPV）DNA检测可作为宫颈癌初筛方法,用于中国及其他发展中国家宫颈癌的二级预防。这一结论来自题为《高危型人乳头瘤病毒DNA检测作为宫颈癌初筛方法的有效性研究——中国以人群为基础的17个筛查研究综合分析》的论文。     

用碱洗脱法检测DNA-蛋白质交联和DNA链间交联

本方法以DNA单链断裂的检测为基础,在背景γ射线照射下进行DNA交联检测。所建方法与Kohn氏原法相比,洗脱时间大为缩短,实验所用主要材料都能立足国内。本文引入“交联度”这个参数,能同时相对定量地表示DNA总交联、DNA-蛋白质交联和DNA链间交联。此外还从DNA、蛋白质两方面确证了DNA-蛋白质交联的存在。     

陈旧家禽血液基因组DNA的快速提取

目的:建立一种从陈旧家禽血液中快速提取基因组DNA的方法。方法:以传统蛋白酶K法为基础进行优化。以陈旧鸡血为实验材料,新鲜抗凝鸡血为对照,经过生理盐水预处理、高浓度SDS裂解液和蛋白酶K消化、酚氯仿抽提后,对产物进行凝胶电泳检测、紫外分光光度检测和PCR扩增。结果:提取时间缩短为5～6h,所得基因组DNA纯度高、产量大,可用于后续分子生物学实验。结论:快速从陈旧家禽血液中提取DNA的方法被成功建立。     

高通量筛选在抗病毒药物筛选中的应用

高通量筛选(high throughput screening, HTS)是以分子和细胞水平的实验方法为基础,在微孔板上以自动化操作系统执行实验过程,通过灵敏快速的检测仪器采集实验数据、运用计算机对实验数据进行实验结果的分析处理.因此HTS具有大量样品的快速筛选、分子和细胞水平的特异性作用靶点、检测系统的高灵敏度、自动化操作系统和数据采集传输处理系统等优点.     

快速检测中药材乌梢蛇LAMP方法的建立

乌梢蛇作为一种名贵中药材,市面上伪品较多,干燥熏黑处理后的样品,更是真伪难辨。本研究致力于开发一套基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)为基础的快速筛查乌梢蛇的方法。本研究以乌梢蛇12srRNA基因序列为基础设计并筛选出1套LAMP引物。通过调整反应条件,建立了对乌梢蛇LAMP的检查方法。结果显示,62℃下连续反应15min左右出现典型的"S"型荧光吸收曲线,实现了对乌梢蛇12srRNA基因序列的特异扩增。根据LAMP灵敏度高的特点,本研究简化了DNA的提取方法,缩短了检测的时间。相对于常规的PCR方法,本研究建立的以快速DNA提取为基础的乌梢蛇LAMP快速筛查方法具有简单、快速、灵敏、对设备要求低等特点,适用于对中药材乌梢蛇的快速筛查。     

New immunoaffinity-LC-MS/MS methodology reveals that Aag null mice are deficient in their ability to clear 1,N6-etheno-deoxyadenosine DNA lesions from lung and liver in vivo

The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine ((epsilon)dA). Previous attempts to determine the in vivo role of Aag in (epsilon)dA repair were complicated by technological difficulties in measuring low levels of (epsilon)dA in genomic DNA. Here we describe the development of a new method for (epsilon)dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of (epsilon)dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal (epsilon)dA bases, these lesions can also be eliminated from the genome via an alternative mechanism.     

Selective Medium for the Isolation of Streptococci from Clinical Specimens

Incorporating neomycin and nalidixic acid into a blood-agar base resulted in a medium highly selective for beta-hemolytic streptococci under conditions in which detection of streptococcal colonies by conventional means would have been very difficult.     

Cost efficiency in the detection of chytridiomycosis using PCR assay

Chytridiomycosis is an emerging infectious disease of amphibians associated with mass mortalities and population declines worldwide. Recent technological advances have resulted in a highly sensitive, non-invasive technique for diagnosing the disease based on a quantitative (real-time) polymerase chain reaction (qPCR) assay. The qPCR assay yields the most accurate and informative data of any available detection technique. However, due to the relatively high costs involved, it has yet to attain widespread use by chytridiomycosis researchers. Using the results of a disease survey of 467 wild frogs from eastern Queensland, Australia, we examine the necessity of triplicate assays in qPCR detection of chytridiomycosis. We describe a singlicate qPCR assay that can be used to substantially decrease costs, with no significant decrease in sensitivity. We also demonstrate that detection of chytridiomycosis by use of the conventional PCR assay may lead to appreciable underestimations in disease prevalence. We recommend that amphibian disease researchers adopt the singlicate qPCR assay as the primary means of chytridiomycosis detection.     

我国生物技术基地平台现状与发展建议

生物技术是21世纪以来新一轮科技革命和产业变革的核心,而生物技术基地平台是开展生物技术研究、生产和服务的载体,是生物技术创新体系的重要组成部分。对世界主要发达国家及我国生物技术基地平台的总体规模和建设情况进行梳理分析,并借鉴国外经验,提出改进我国生物技术基地平台建设的政策建议,以供参考。     

Chemical cleavage of DNA with single base mismatches for detection of mutations of unknown localization

The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.     

The ethnoecology of maize variety management: A case study from Mexico

This paper presents a case study of the relationship between farmers' knowledge of maize varieties and their selection and management of these varieties under conditions of technological change. Research for this paper was done among Spanish-speaking small farmers in an ejidoof central Chiapas, Mexico. This ejido is well integrated into the market, and the use of modem technologies is widespread. This research demonstrates that farmers have an extensive and widely shared knowledge of their maize varieties. This knowledge reflects objective maize characteristics. Variation occurs in the farmers' selection and management of maize varieties, but on average the variation deviates from a random pattern in the direction predicted by the farmers' knowledge base. They have incorporated the technological changes brought about by development into their knowledge base. Farmers maintain maize varieties with contrasting traits, and their knowledge base provides important information about which traits and constraints are important to them.     

Nanotechnologies for pathogen detection: Future alternatives?

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies.Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.     

New cyanine dyes as base surrogates in PNA: forced intercalation probes (FIT-probes) for homogeneous SNP detection

Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye-PNA-conjugates, the YO-containing PNA has properties useful for homogeneous SNP detection. YO-PNA is demonstrated to signal the presence of fully complementary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single base mutations at non-stringent conditions.     

Detection of single-base DNA mutations by enzyme-amplified electronic transduction

Here we describe a method for the sensitive detection of a single-base mutation in DNA. We assembled a primer thiolated oligonucleotide, complementary to the target DNA as far as one base before the mutation site, on an electrode or a gold-quartz piezoelectric crystal. After hybridizing the target DNA, normal or mutant, with the sensing oligonucleotide, the resulting assembly is reacted with the biotinylated nucleotide, complementary to the mutation site, in the presence of polymerase. The labeled nucleotide is coupled only to the double-stranded assembly that includes the mutant site. Subsequent binding of avidin-alkaline phosphatase to the assembly, and the biocatalyzed precipitation of an insoluble product on the transducer, provides a means to confirm and amplify detection of the mutant. Faradaic impedance spectroscopy and microgravimetric quartz-crystal microbalance analyses were employed for electronic detection of single-base mutants. The lower limit of sensitivity for the detection of the mutant DNA is 1 x 10-14 mol/ml. We applied the method for the analysis of polymorphic blood samples that include the Tay-Sachs genetic disorder. The sensitivity of the method enables the quantitative analysis of the mutant with no PCR pre-amplification.     

麻黄生物总碱提取生产工艺改进

设计考察了一种新的麻黄生物碱提取生产工艺,旨在解决现生产中的污染问题。在碱性条件下将麻黄生物碱盐苛化为生物碱形式,直接用二甲苯提取后,用2％草酸将生物碱转化为草酸盐萃取,二甲苯在工厂原有设备基础上回收循环利用,在解决污水问题的同时,简化工艺。采用析因试验确定最佳提取条件,包括提取温度、加饱和氢氧化钠量、麻黄草的粉碎程度。并考查了提取时间对提取率的影响;建立测定提取液中生物碱含量的方法。