Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations

We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.     

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.     

A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis ( Map ) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case–control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map -negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance ( P  < 5 × 10⁻⁵). Two regions, one on chromosome 3 (near EDN2 ) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively ( P  <   5 × 10⁻⁷, genome-wide Bonferonni P  <   0.05).     

两种高危型HPV检测技术诊断宫颈病变的临床价值比较

目的:比较酶切信号放大法(Cervista)与导流杂交基因芯片技术(Hybri Ma)检测高危型人乳头状瘤病毒(HR-HPV)诊断宫颈上皮内瘤变2级或2级以上(CIN2+)的临床价值。方法:随机选择288例2012年3月至2013年1月在哈尔滨医科大学附属第一医院妇科门诊进行新柏氏液基细胞学检查的年龄在20~65岁的宫颈细胞学检测未明确意义的不典型鳞状细胞(ASCUS)的患者,采用Cervista技术与Hybri Ma技术进行高危型HPV检测,并对入组的患者行阴道镜下宫颈活组织检查。以病理学诊断结果为金标准,比较Cervista技术与Hybri Ma技术诊断宫颈上皮内瘤变2级或2级以上(CIN2+)的敏感度、特异度及ROC曲线。结果:在入组的288例患者中,Cervista技术和Hybri Ma技术检出高危型HPV的阳性率分别为49.31%和51.39%(P0.05),其诊断CIN2+的敏感度分别为95.65%和91.30%(P0.05),特异度分别为59.50%和56.20%(P0.05),阳性预计值分别为30.99%和28.38%(P0.05),阴性预计值分别为98.63%和97.14%(P0.05)。两组ROC曲线下面积分别为0.776和0.738(P0.05)。结论:Cervista技术与Hybri Ma技术诊断CIN2+的临床价值相当。     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

转基因植物检测技术的研究进展

现代植物基因工程使转基因植物及其产品越来越多地进入人们的生活,转基因植物安全性在世界范围内引起了广泛关注,对转基因植物检测技术的需求也越来越紧迫。就转基因植物检测技术的研究进展进行综述,重点介绍以基因和蛋白为目标的检测技术,包括PCR、ELISA和基因芯片技术的最新进展,并对不同方法的优缺点进行对比。此外,提出对特定代谢产物的检测是转基因植物检测的重要组成部分,是以后检测技术的发展趋势之一。最后,以差异蛋白为检测目标,结合研究工作提出基于双向电泳技术的转基因植物检测方法及其产品溯源方案。     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

基于微流控芯片的温度梯度毛细管电泳检测大肠癌石蜡标本中K-ras基因突变

K-ras基因突变检测可用于大肠癌的早期筛查与诊断,并有利于筛选出抗表皮生长因子受体靶向药物治疗有效的大肠癌患者,以实现肿瘤的个体化治疗．采用以倾斜式热辐射原理建立的微流控温度梯度毛细管电泳(temperature gradient capillary electrophoresis,TGCE)基因突变检测系统,实现了对98例石蜡包埋大肠癌组织中K-ras基因突变的高灵敏度筛查,突变阳性检出率为47.96%,显著高于PCR产物直接测序的23.47%．克隆测序显示该方法至少能检测到2.08%的K-ras基因突变体．K-ras基因突变与临床病理学参数的关系分析显示,直肠癌中K-ras基因突变率明显高于结肠癌(P < 0.05),而与年龄、性别、组织学类型和肿瘤分期等无显著相关性．该检测方法为肿瘤早期诊断和指导临床用药提供了一种灵敏度高、检测速度快、便于大规模筛查的有效手段.     

微流控芯片电泳分离血清高密度脂蛋白亚类的研究

描述了一种微流控芯片电泳快速分离血清高密度脂蛋白(high density lipoprotein,HDL)亚类的方法.利用自制的微流控芯片,结合激光诱导荧光检测系统,40mmol/L Tricine、50mmol/L甲基葡胺(MEG)、0.2mmol/LSDS(pH8.5)为样品缓冲液,40mmol/L Tricine、50mmol/LMEG、0.01mmol/L SDS(pH8.5)为分离缓冲液,4min内HDL3和HDL2两种亚类得到基线分离.该法操作过程简单,重复性较佳,测试费用低廉,在临床HDL亚类的检测中具有较好的应用前景.