Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.
Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.
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DOI：
10.1016/j.ab.2015.08.015
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发表时间：
2015-11-15
影响因子：
2.9
通讯作者：
Benner SA
中科院分区：
文献类型：
--
作者：
Glushakova LG;Sharma N;Hoshika S;Bradley AC;Bradley KM;Yang Z;Benner SA
核酸（NA）靶向检测和定量病毒DNA和RNA（统称xNA），以支持流行病学监测，并在个体患者中指导治疗。它们通常使用聚合酶链反应（PCR）和逆转录PCR。虽然这些都有快速周转，他们是昂贵的运行。多重化将允许它们的成本分散在多个靶标上，但通常仅具有较低的灵敏度和准确性、噪声、假阳性和假阴性;这些是由多重化试剂盒中的多个核酸引物和探针之间的相互作用引起的。在这里，我们提供了一种用于一组呼吸道病毒的多重检测方法，该方法通过结合来自合成生物学新兴领域的几种核酸类似物来缓解这些问题：（i）自避免分子识别系统（SAMRS），其有助于多重检测，以及（ii）人工扩增遗传信息系统（AEGIS），其能够实现低噪声PCR。这些都是由“音译”技术补充，该技术将目标中的标准核苷酸转化为产物中的AEGIS核苷酸，从而改善杂交。该组合支持基于Luminex的多重呼吸道检测板，该检测板可区分流感病毒A和B、呼吸道合胞病毒、严重急性呼吸综合征冠状病毒（SARS）和中东呼吸综合征（MERS）冠状病毒，在20 μl样本中检测到少至10个MERS病毒粒子。
Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by “transliteration” technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample.