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发表于 2011-4-13 11:42 |只看该作者 |倒序浏览 |打印
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<http://www.sciencedaily.com/releases/2011/04/110411103719.htm>

New Technique Tracks Viral Infections, Aids Development of Antiviral Drugs

ScienceDaily (Apr. 11, 2011) — Scientists at the Naval Research
Laboratory Center for Bio-Molecular Science and Engineering have developed
a method to detect the presence of viruses in cells and to study their
growth. Targeting a virus that has ribonucleic acid (RNA) as its genetic
makeup, the new technique referred to as locked nucleic acid (LNA) flow
cytometry-fluorescence in situ hybridization (flow-FISH), involves the
binding of an LNA probe to viral RNA.

While individual parts of the technique have been developed previously,
Drs. Kelly Robertson and Eddie Chang, in collaboration with researchers at
the NRL Lab for Biosensors and Biomaterials, demonstrate for the first time
that the combination of LNA probes with flow-FISH can be used to quantify
viral RNA in infected cells. This also allows the scientists to monitor the
changes in viral RNA accompanying antiviral drug treatment.

Once the probe is bound to the viral RNA inside mammalian cells, it is
tagged with a fluorescent dye, then thousands of these tagged cells are
measured rapidly by "flow cytometry" -- a method for counting and examining
microscopic particles, such as cells and chromosomes, by suspending them in
a stream of fluid and passing them by an electronic detection apparatus.

"The ability to rapidly measure thousands of cells for the presence of
virus, sets this technique apart from currently used methods to monitor
viral replication," said Robertson.

Traditionally, antibodies used to detect viruses must be produced and
calibrated for each specific strain and are highly susceptible to viral
mutations. Assays commonly used for quantifying viral loads and for drug
development can be time consuming and rely on visible signs of cell damage,
which is not produced in all viruses and can take long periods of time to
occur.

Techniques such as quantitative reverse transcription-polymerase chain
reaction (qRT-PCR), microarrays, and enzyme-linked immunosorbent assays
(ELISAs), while highly sensitive, involve the lysis [the breaking down] of
cells prior to measurement and are therefore unable to provide information
about cellular viability, infected cell phenotypes, percentage of infected
cells or the variation in infection among a cell population. The LNA probe
differs from traditional nucleotide probes by binding more tightly to its
target RNA.

LNA-flow FISH presents a fast and easy way to screen for compounds with
antiviral activity and could be adapted for monitoring infections in the
blood for vaccine therapy and development. This method adds a necessary
tool for several emerging areas in cell biology that enables the use of
high throughput measurements for entire populations and improves
statistical analyses.

"This method can be expanded by adding more than one kind of LNA probe to
enable multiple detection of different viral and host RNA," adds Robertson.
"The multiplexing enhancement can be used to better understand infectious
agents, allowing this technique to be used to aid in the development of
antiviral drugs for a variety of viruses."

LNA flow-FISH offers an advantage over other techniques due to its
simplicity and superiority. Methods involving genetic recombination of the
virus to express a fluorescent protein as a means to mark the presence of
virus can utilize flow cytometry for large-batch analysis of infected
cells. However, an exception to this approach is viral strains that have
not acquired genetic mutations, known as wild-type viruses (such as strains
of Human Immunodeficiency Virus-HIV), which would require a large initial
investment of labor for engineering each virus of interest.



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发表于 2011-4-13 11:43 |只看该作者
新技术曲目病毒感染,艾滋病的抗病毒药物开发

科学日报(2011年4月11号) - 海军研究的科学家在
实验室生物分子科学与工程研究中心已经开发
一个方法来检测细胞中的病毒的存在,并研究其
增长。靶向一种病毒,其基因具有核糖核酸(RNA)的
化妆,这项新技术被称为锁核酸(LNA)的流
流式细胞仪,荧光原位杂交(流鱼),涉及
绑定一个LNA探针病毒的RNA。

虽然该技术的各个部分已经制定以前,
博士。凯利罗伯逊和研究人员的合作与埃迪常在
生物传感器和生物材料的夺标实验室,首次展示
认为与流动相结合鱼的LNA探针可用于量化
病毒RNA在感染的细胞。这也使得科学家们能够监测
在病毒RNA的变化伴随抗病毒药物治疗。

一旦探针结合在哺乳动物细胞的病毒RNA,这是
这些标记细胞标记用荧光染料,然后数千
测量迅速被“流式细胞仪” - 一个用于计数和检查方法
微观粒子,如细胞和染色体,它们在通过暂停
一流体流经过他们的电子检测仪器。

“能够迅速衡量是否存在成千上万的细胞
病毒,设置从目前这种技术使用的方法除了监测
病毒的复制,“罗伯逊说。

传统上,抗体用于检测病毒,并须出示
校准为每个特定菌株,并极易受病毒
突变。常用的定量检测病毒载量和药物
发展可能会非常耗时和依靠细胞损伤的明显迹象,
这是不生产的所有病毒和所花费的时间长的期间内
发生。

技术如定量逆转录聚合酶链
反应(qRT - PCR)技术,微阵列和酶联免疫吸附法
(ELISA试剂盒),而高度敏感,涉及到溶解的[向下突破]
测量之前和细胞,因此无法提供信息
关于细胞的活力,受感染的细胞表型,受感染的百分比
在感染的细胞或细胞群中一个变化。 LNA的探头
从传统的核苷酸探针结合更紧密的不同之处,就其
靶RNA。

低噪声放大器流鱼呈现快速简便的方法来筛选的化合物
抗病毒活性,可用于监测在感染改编
血液疗法和疫苗的发展。这种方法增加了一个必要的
几个新兴的细胞生物学方面的工具,使得使用
为全体人民,提高高通量测量
统计分析。

“这种方法可以通过增加多个低噪声放大器探头那种
让不同的RNA病毒与宿主多种检测,补充说:“罗伯逊。
“复用提高可用于更好地了解传染病
剂,使这种技术被用来在发展援助
对多种病毒的抗病毒的药物。“

低噪声放大器流鱼类提供了比其他技术优势,由于其
简单性和优越性。方法涉及的基因重组
病毒荧光蛋白表达的一种手段,以纪念在场
病毒可以利用流动的大批量感染流式细胞仪分析
细胞。但是,这种方法的例外是,有病毒株
没有获得基因突变,为野生型病毒,已知的(如株
人类免疫缺陷病毒HIV)的,这需要大量的初始
劳动力投资工程每个感兴趣的病毒。

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发表于 2011-4-13 21:54 |只看该作者
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