Real-time fluorescent quantitative immuno-PCR method for determination of fluoranthene in water samples with a molecular beacon

A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.

Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages

Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis(MTB). Once the pathogen enters the body, it subverts the host immune defenses and thrives for extended periods of time within the host macrophages in the lung granulomas, a condition called latent tuberculosis(LTB). Persons with LTB are prone to reactivation of the disease when the body's immunity is compromised. Currently there are no reliable and effective diagnosis and treatment options for LTB, which necessitates new research in this area. The mycobacterial proteins and genes mediating the adaptive responses inside the macrophage is largely yet to be determined. Recently, it has been shown that the mce operon genes are critical for host cell invasion by the mycobacterium and for establishing a persistent infection in both in vitro and in mouse models of tuberculosis. The Yrb E and Mce proteins which are encoded by the MTB mce operons display high degrees of homology to the permeases and the surface binding protein of the ABC transports, respectively. Similarities in structure and cell surface location impute a role in cell invasion at cholesterol rich regions and immunomodulation. The mce4 operon is also thought to encode a cholesterol transport system that enables the mycobacterium to derive both energy and carbon from the host membrane lipids and possibly generating virulence mediating metabolites, thus enabling the bacteria in its long term survival within the granuloma. Various deletion mutation studies involving individual or whole mce operon genes have shown to be conferring varying degrees of attenuation of infectivity or at times hypervirulence to the host MTB, with the deletion of mce4 A operon gene conferring the greatest degree of attenuation of virulence. Antisense technology using synthetic si RNAs has been used in knocking down genes in bacteria and over the years this has evolvedinto a powerful tool for elucidating the roles of various genes mediating infectivity and survival in mycobacteria. Molecular beacons are a newer class of antisense RNA tagged with a fluorophore/quencher pair and their use for in vivo detection and knockdown of mR NA is rapidly gaining popularity.

REAL-TIME DETECTION OF SURVIVIN mRNA EXPRESSION IN CERVICAL CANCER CELL LINES USING MOLECULAR BEACON IMAGING

Objective To detect the expression of survivin mRNA in cervical cancer cell lines using molecular beacon imaging technology. Methods Human cervical cancer cells (HeLa and SiHa) and human fetal lung fibroblast HFL-I were cultured in vitro. After adding 100nmol/L survivin mRNA molecular beacon, the fluorescent signals were observed under fluorescent microscope. The expressions of survivin in cervical cancer cells and HFL-I cell were examined by immunocytochemical streptravidin-biothin peroxidase (SP) assay at the same time. Results Two kinds of survivin mRNA molecular beacon, with different color fluorescence, had strong fluorescent signal in cervical cancer cell lines, and the signal in SiHa cell line was stronger, but these signals were not found in HFL-I ; Immunocytochemical staining of positive survivin was located in the cytoplasm of cervical cancer cell lines HeLa and SiHa, whereas, no expression of survivin was detected in HFL-I cell line. Conclusion The technology of molecular beacon imaging can be used to detect the expression of survivin mRNA in viable cells successfully, and may provide a new approach to the diagnosis of early stage cervical cancer and the following-up in the clinic.

Real-time Monitoring of Nucleotide Excision of Molecular Beacon Catalyzed by Klenow Fragment

Klenow fragment（KF） uses the activity of a separate exonuclease to excise nucleotide, which is a crucial step in DNA replication and repair. Here is a novel sensitive and convenient method introduced for real-time monitoring nucleotide excision by KF with a molecular beacon as a detecting probe in a homogeneous solution. This method, which overcomes the drawbacks of traditional methods such as discontinuity, time consuming and low sensitivity, was used to assay KF activity and the detection limit reached up to 0.4 U/mL. In addition, the method was applied to investigating the effects of metal ions and chemical drugs on the reaction. The results demonstrate that it is a potential high-throughput assay for screening inhibitors and activity analysis of KF in vitro.

奥曲肽修饰的壳聚糖纳米递送分子信标成像肺癌研究

目的 探究通过奥曲肽(OCT)修饰的壳聚糖(CS)miR-155分子信标(miR-155-MB)纳米(CS-miR-155-MB-OCT)识别miR-155并成像肺癌细胞用于肺癌早期诊断。方法 通过尾静脉注射A549肺癌细胞建立肺移植瘤裸鼠模型;通过鼻腔滴入Cre腺病毒,分别在滴入腺病毒后的4、6、8、12周处死小鼠建立LSL K-ras G12D转基因小鼠肺部的非典型增生、腺瘤、原位癌、肺腺癌的不同病变时期肺腺癌模型;取肺组织行HE染色观察。免疫组化染色检测肺移植瘤组织及不同病变时期转基因小鼠肺组织生长抑素受体2(SSTR2)的表达;实时荧光定量PCR检测不同病变时期转基因小鼠肺组织miR-155的表达。在肺移植瘤裸鼠模型中,尾静脉注射CS-miR-155-MB或CS-miR-155-MB-OCT;转基因小鼠模型中,尾静脉注射CS-miR-155-MB-OCT;活体成像仪检测肺移植瘤裸鼠及转基因小鼠不同病变时期肺部荧光信号;制作肺组织冰冻切片,激光共聚焦扫描显微镜(CLSM)检测荧光信号来源。结果 HE染色显示成功构建了肺移植瘤裸鼠模型及转基因小鼠肺部的非典型增生、腺瘤、原位癌、肺腺癌的不同病变时期肺癌模型。肺移植瘤及不同病变时期病变组织均表达SSTR2。转基因小鼠模型中,随着疾病进展,miR-155表达逐渐升高(P<0.05)。在肺移植瘤裸鼠模型中,CS-miR-155-MB-OCT组肺部荧光信号强于CS-miR-155-MB组(P<0.05);转基因小鼠模型中,随着肺癌的进展,荧光信号逐渐增强(P<0.05);将肺组织再次成像后发现荧光信号来自肺部,CLSM发现荧光信号来自肿瘤细胞及部分正常肺泡上皮细胞。结论 CS-miR-155-MB-OCT能够根据产生的荧光强度变化动态反映肺癌的发生发展,从而为肺癌的早期诊断提供新技术。

基于DNAzyme的荧光生物传感器快速检测金黄色葡萄球菌研究

【目的】为解决传统方法检测金黄色葡萄球菌耗时长、操作复杂等问题,研发一种基于脱氧核酶(DNAzyme)的荧光生物传感器,实现金黄色葡萄球菌的快速检测。【方法】将特异性DNAzyme和互补链Subatrate相结合制成荧光生物传感器,并对荧光生物传感器进行生物材料浓度和溶液pH优化,并对金黄色葡萄球菌、大肠埃希菌、芽孢杆菌、无乳链球菌、变形杆菌等进行特异性检测;最后对牛奶样品进行基于DNAzyme的荧光生物传感器的试验验证。【结果】基于DNAzyme的荧光生物传感器在pH为6.8时,3 min内可以实现对金黄色葡萄球菌的检测,线性范围为1~1×10^(7) cfu·mL^(−1),最低检测限为1 cfu·mL^(−1)。【结论】基于DNAzyme的荧光生物传感器解决了传统检测方法耗时长、操作复杂等问题,实现了对金黄色葡萄球菌的快速检测,具有重要的应用价值。

Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA

Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.

基于分子信标技术的肉类食品动物源性成分多重筛检方法

建立一种基于通用型分子信标检测及探针熔解曲线分析技术的肉类食品动物源性成分多重筛检方法。通过对猪、牛、羊等9种常见食用畜禽动物基因组多序列比对及生物信息学分析,选取线粒体基因组16s r DNA中具有特定结构的基因片段作为检测靶序列,设计单一的通用型引物和长链分子信标。以标准基因重组载体及实体样品基因组为检测对象,确定各动物源性成分特征退火温度(Tm),建立基于实时荧光PCR及熔解曲线分析的标准检测方法,并对筛检方法的灵敏度和特异性进行考察。应用所建立检测方法,9种畜禽动物基因组扩增产物单一,测序结果与参考序列一致。对梯度稀释的猪标准基因重组载体检测发现,终浓度10~8~10~1copies/μL模板扩增Ct值范围为15~36个循环(回归系数R~2=0.99),熔解峰特征Tm为(68.0±0.1)℃。对9类畜禽肉类标准基因重组载体和实体样品进行独立检测,各类动物源性基因均形成可辨识的特异性熔解峰(Tm分别为猪68.0℃、牛64.3℃、羊65.1℃、驴65.8℃、马63.4℃、驼60.6℃、狗61.8℃、鸡52.6℃、鸭56.7℃),混合样品检测可见独立的种属特征熔解峰或融合形成新熔解峰。基于通用型引物及分子信标的实时荧光PCR检测方法具有理想的灵敏度和特异性,可实现对常见畜禽肉类食品源性成分的单管多重鉴定,在肉类食品源性成分筛检方面具有一定的推广应用价值。

旅行商问题的DNA可视化计算模型

基于“DNA折纸术”提出一个旅行商问题的解决方案,利用“DNA折纸术”折叠出固定大小的DNA纳米结构作为DNA折纸基底,利用分子信标表示旅行商问题中的城市(即顶点)和路径,将旅行商问题的路径映射为一个有向图,选择根节点最终将问题映射为有向树,并将有向树锚定在DNA折纸基底上,利用杂交链式反应反应出经过每个点且长度最短的DNA长链,即为该问题的最优解,同时用荧光标记的分子信标个数检测其路径长度,实现求解旅行商问题的可视化.通过实例模拟和仿真实验验证方法的有效性和可行性,分析给出该DNA可视化计算模型的复杂度.

TaqMan-分子灯标:一种新型的荧光基因检测探针

在TaqMan及分子灯标的基础上开发了一类新型的均相荧光检测探针———TaqMan 分子灯标 (TaqMan MB) ,该探针集合了分子灯标的发夹结构及TaqMan探针降解作用的工作原理 ,使检测效果更好 .与实时PCR仪联用 ,可用于靶基因的定量检测 .

一种新型荧光探针——分子信标的研究及应用进展

分子信标是一种基于荧光能量转移原理而设计的发夹型寡聚核酸荧光探针。它通过与核酸等靶分子相互作用后发生构象的变化而产生荧光信号 ,对靶分子的检测具有灵敏度高、选择性强、适合于活体实时检测等优点。目前已广泛应用于生物化学分析、生物医学研究和环境监测等各领域。本文对分子信标的设计原理及其研究和应用进展进行了综述。

一种基于分子信标荧光探针快速检测烟草花叶病毒的新方法

研究了一种利用新型荧光探针———分子信标进行植物病毒检测的方法 ,可以不要求对病毒RNA进行严格的分离和纯化 ,就能快捷地得到检测结果。此方法被用于烟草花叶病毒 (tobaccomosaicvirus,TMV)基因组RNA的检测 。

基于竞争触发滚环扩增的荧光适配体传感器高灵敏检测凝血酶

利用目标蛋白、适体探针、挂锁探针和与适体探针匹配的互补序列之间的竞争反应,发展了一种基于滚环扩增的高灵敏荧光适配体传感器。在不含目标蛋白时,互补序列由于与适体探针杂交形成双链,因此不能与挂锁探针杂交以触发连接-滚环放大反应。相反,有目标蛋白存在时,由于目标蛋白与适体探针结合,使得互补序列被置换下来,从而可以与挂锁探针杂交。在DNA连接酶的作用下,挂锁探针被进一步环化并在Phi 29 DNA聚合酶的作用下发生滚环扩增反应。扩增产物含有许多能与分子信标检测探针环状部分杂交的重复序列,从而分子信标被打开产生荧光信号。对互补序列的长度及挂锁探针的浓度进行了考察。在优化的实验条件下,本传感系统可以实现对的凝血酶高灵敏检测,线性范围为0.067~32 nmol/L,检出限为0.03 nmol/L（约90 amol目标分子）。通过设计不同的适体探针和相关核酸序列,本传感系统可以作为一种通用型方法用于其它目标物的分析。

分子信标荧光定量PCR快速检测结核分枝杆菌方法的初步研究

目的建立分子信标荧光定量PCR快速检测结核杆菌方法,为临床结核病的诊断提供特异性强、灵敏度高、可标准化、自动化的检测方法。方法根据GenBank数据库公布的结核分枝杆菌IS6110基因的保守序列,设计引物与分子信标探针,建立结核分枝杆菌荧光定量PCR检测方法;以10种细菌作对照分析该方法的特异性与灵敏度;应用已建立的方法检测结核分枝杆菌,评价其应用价值。结果所建立的分子信标荧光定量PCR检测10种细菌,只有结核分枝杆菌有荧光信号,与其他细菌无交叉反应,4拷贝/PCR反应体系的结核分枝杆菌都能有效检出。对100株结核分枝杆菌的检测结果为阳性,单一检测时间仅需2h。结论分子信标荧光定量PCR检测方法快速、灵敏度高、特异性强,可用于快速诊断结核病,为结核病的诊断与治疗提供新的检测手段。

改良分子信标-实时PCR快速检测副溶血弧菌

目的 :建立改良分子信标 -实时 PCR检测副溶血弧菌的快速方法 ,应用于副溶血弧菌食物中毒的快速诊断和海产品检验。方法 :根据 Gen Bank公布副溶血弧菌的耐热直接溶血毒素基因 (TDH)的保守序列 ,设计一对引物和改良分子信标探针 ,用 FAM荧光剂标记探针的 5′,并进行特异性和灵敏度分析 ;同时以 11种细菌作对照 ,建立改良分子信标检测副溶血弧菌的实时 PCR反应体系 ,应用于副溶血弧菌食物中毒快速诊断和食品微生物检测。结果 :检测 12种细菌 ,只有副溶血弧菌有荧光信号 ,与其他细菌无交叉反应 ,DNA灵敏度为 16 6 .6 fg/ μl,菌液灵敏度为 6 9cfu/ ml或 6 cfu/ PCR反应体系。改良分子信标 -实时 PCR反应体系检测 4 0株副溶血弧菌均出现特异的荧光信号 ,无干扰。对 3起细菌性食物中毒共 4 8份样品和 10 0份海产品进行检测 ,9份副溶血弧菌实时 PCR阳性 ,其中 7份副溶血弧菌细菌培养阳性 ,其余样品都为阴性。检测时间仅需 2 h。结论 :改良分子信标 -实时 PCR检测体系快速、灵敏度高 ,特异性强 ,可用于副溶血弧菌食物中毒的快速诊断 。

用于分子识别的荧光标记探针的研究进展

利用荧光标记物进行分子识别是目前生命科学研究的热点课题。比较了几种用于分子识别的荧光标记探针的特性,如有机荧光染料、分子灯塔、纳米半导体量子点以及荧光蛋白探针。并分析了荧光标记探针的发展趋势,指出了理想的荧光标记探针应该具备的特征。

可满足问题的分子信标计算模型(英文)

分子信标(Molecular Beacon)是一种发夹状的荧光探针,它可以特异地和那些与分子信标的环(Loop)互补的核酸靶序列杂交,具有单个碱基错配的检测能力.肽核酸(Peptide Nucleic Acid)是人工合成的核酸(DNA)的类似物.PNA骨架为酰胺键,与DNA补链杂交更稳定,可以阻止聚合酶延伸反应.文中将可满足问题的约束变量编码于分子信标的环部识别区,通过分子信标与使得给定范式为真的变量的PNA补链杂交,再利用PNA链可以阻止聚合酶延伸反应的性质,用限制性内切酶EcoRI降解对应于非解的分子信标,最后通过加热表面使分子信标构形发生变化,产生荧光读解.提出的可满足问题的分子信标计算模型具有可靠性高、无需观察和记录计算的中间结果、读解简单等优点.

基于分子信标的DNA计算

DNA计算是解决一类难以计算问题的一种新方法,这种计算随着问题的增大可以至指数增长.迄今为止,许多研究成果已经成功地提高了它的性能和增加了它的可行性,本文在基于表面的DNA计算中采用了分子信标编码策略,并对分子信标在与对应的补链杂交形成双键时的受力进行分析,给出3—SAT问题的另一种解法.这种方法比现有的方法更有效,更具发展前景.因为它具有编码简单;耗材底;操作时间短;技术先进等优点.本文尝试了分子生物学,光学和力学的结合.这一工作为DNA计算能解决NP-完全问题提供了更有力的依据.

分子信标荧光探针用于抑癌基因ING1表达产物的定量测定

根据抑癌基因 ING1基因的序列设计并合成了检测 ING1转录产物的分子信标核酸探针 ,发展了一种快速测量从正常细胞系和鼻咽癌肿瘤细胞系提取的 ING1转录产物的方法 ,所得结果与用逆转录结合 PCR法 ( RT-PCR)得到的结果相吻合 .并且 ,将能表达 ING1基因的质粒转入肿瘤细胞进行培养后 ,再将分子信标转入肿瘤细胞 ,发现转导了质粒的肿瘤细胞比未转导质粒的肿瘤细胞内的荧光明显增强 ,从而进一步证实了所设计的分子信标核酸探针与

分子信标荧光定量PCR技术检测结核分枝杆菌方法建立及其临床应用

目的建立结核分枝杆菌的分子信标荧光定量PCR检测方法,探讨该方法检测结核分枝杆菌的临床应用价值。方法针对结核分枝杆菌Ag85BDNA特异保守序列设计引物和分子信标探针,并构建标准品。采用分子信标荧光定量PCR法、抗酸染色法、集菌培养法检测158例肺结核患者痰液标本,比较3种方法的检出率。结果所建立方法最低检测限度为2个基因拷贝/反应,在2×102～2×108基因拷贝/mL范围内,Ct值同DNA量的对数呈线性关系。该方法检测5株非分枝杆菌和6株非结核分枝杆菌结果均为阴性,检测结核分枝杆菌H37Rv出现75 bp特异条带。分子信标荧光定量PCR法、抗酸染色法、集菌培养法阳性检出率分别为60.13%、16.46%、31.01%,荧光定量PCR法检出率明显高于抗酸染色法和集菌培养法。结论分子信标荧光定量PCR法具有较高的灵敏度和特异度,对结核病的诊断有一定的临床意义。