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Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA
Analyst ( IF 3.6 ) Pub Date : 2017-08-14 00:00:00 , DOI: 10.1039/c7an01023k Gabriel Antonio S. Minero 1, 2, 3, 4, 5 , Catarina Nogueira 5, 6, 7 , Giovanni Rizzi 1, 2, 3, 4, 5 , Bo Tian 8, 9, 10, 11, 12 , Jeppe Fock 1, 2, 3, 4, 5 , Marco Donolato 5, 6, 7 , Mattias Strömberg 8, 9, 10, 11, 12 , Mikkel F. Hansen 1, 2, 3, 4, 5
Analyst ( IF 3.6 ) Pub Date : 2017-08-14 00:00:00 , DOI: 10.1039/c7an01023k Gabriel Antonio S. Minero 1, 2, 3, 4, 5 , Catarina Nogueira 5, 6, 7 , Giovanni Rizzi 1, 2, 3, 4, 5 , Bo Tian 8, 9, 10, 11, 12 , Jeppe Fock 1, 2, 3, 4, 5 , Marco Donolato 5, 6, 7 , Mattias Strömberg 8, 9, 10, 11, 12 , Mikkel F. Hansen 1, 2, 3, 4, 5
Affiliation
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We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.
中文翻译：
实时光磁检测登革热血清型2合成DNA的LAMP扩增子的序列特异性验证
我们报告了一种优化的光磁技术，可使用两种不同的检测方法在理想和非理想实验室条件下对登革热病毒进行实时分子检测。第一种方法是基于对附着于生物素化LAMP扩增子的链霉亲和素包被的磁性纳米颗粒的流体动力学体积的检测。我们证明了在20分钟内在理想的无污染实验室环境中检测亚飞摩尔登革热DNA靶标的浓度。第二种检测方法基于功能化磁性纳米粒子与LAMP扩增子环的序列特异性结合。熔解研究表明，真正的阳性和伪扩增子具有不同的熔点，这使我们能够区分它们。发现这与随后的关于LAMP扩增子的实时序列特异性鉴别的研究很好地吻合。特异性结合导致磁性纳米粒子聚集通过与在LAMP延伸阶段出现的多个位点（环）结合。纳米团簇的形成通过游离纳米颗粒引起的光信号的耗尽来监测。经过序列特异性验证后，我们要求在20分钟的LAMP污染背景下检测到低至100 fM的登革热靶标。
更新日期：2017-09-08
中文翻译：
实时光磁检测登革热血清型2合成DNA的LAMP扩增子的序列特异性验证
我们报告了一种优化的光磁技术，可使用两种不同的检测方法在理想和非理想实验室条件下对登革热病毒进行实时分子检测。第一种方法是基于对附着于生物素化LAMP扩增子的链霉亲和素包被的磁性纳米颗粒的流体动力学体积的检测。我们证明了在20分钟内在理想的无污染实验室环境中检测亚飞摩尔登革热DNA靶标的浓度。第二种检测方法基于功能化磁性纳米粒子与LAMP扩增子环的序列特异性结合。熔解研究表明，真正的阳性和伪扩增子具有不同的熔点，这使我们能够区分它们。发现这与随后的关于LAMP扩增子的实时序列特异性鉴别的研究很好地吻合。特异性结合导致磁性纳米粒子聚集通过与在LAMP延伸阶段出现的多个位点（环）结合。纳米团簇的形成通过游离纳米颗粒引起的光信号的耗尽来监测。经过序列特异性验证后，我们要求在20分钟的LAMP污染背景下检测到低至100 fM的登革热靶标。
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