Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.
Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.
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DOI：
10.1371/journal.pntd.0009114
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发表时间：
2021-01
影响因子：
3.8
通讯作者：
Stoecker K
中科院分区：
文献类型：
--
作者：
Müller K;Daßen S;Holowachuk S;Zwirglmaier K;Stehr J;Buersgens F;Ullerich L;Stoecker K
分子诊断学在许多传染病和被忽视的疾病的鉴定中已经成为必不可少的，而核酸的检测往往是大多数传染病病原体的金标准技术。然而，已建立的技术，如聚合酶链式反应(PCR)，是耗时的实验室限制技术，而快速测试，如侧流免疫层析测试，往往缺乏所需的灵敏度和/或特异性。在这里，我们提出了一种负担得起的、高流动性的替代方法，用于使用脉冲控制扩增(PCA)快速鉴定感染性病原体。PCA是新一代核酸放大技术，它使用快速能量脉冲来加热微循环器(直接嵌入扩增反应中的微型金属加热元件)几微秒，从而只加热反应体积的一小部分。加热的微循环器几乎瞬间冷却，导致超快的加热和冷却循环，在此期间发生经典的靶序列扩增。这将总放大时间减少了高达10倍，在小型便携式原型设备上运行时，只需15分钟即可实现从样品到结果的工作流程。在这项原理证明研究中，我们设计了一种检测鼠疫耶尔森氏菌的主成分分析方法，以证明这项技术的有效性。观察到的检出限分别为434个拷贝/反应(纯化DNA)和35个细胞/反应(粗品)。PCA提供快速和分散的分子诊断，适用于任何需要快速、现场检测感染性病原体的地方，即使在资源有限的条件下也是如此。它结合了聚合酶链式反应的敏感性和特异性，以及迄今存在的快速检测的快速和简单。快速和可靠的现场诊断是应对传染病暴发和抗击被忽视的热带病的重要组成部分。在这方面，分子诊断学代表着当前的黄金标准。然而，目前绝大多数的分子诊断方法(如聚合酶链式反应)都是受实验室限制的技术，需要昂贵而繁重的仪器，而且执行起来很耗时。此外，在资源有限、基础设施很少或根本没有基础设施的国家，样本往往必须长途运输到少数几个能够进行此类诊断的参考实验室。这大大减慢了整个诊断过程，从而大大推迟了控制疫情和启动医疗对策所必需的重要决定。到目前为止，对有限数量的感染性病原体只有很少的快速现场诊断测试，而且它们往往缺乏敏感性和/或特异性。在这里，我们提出了一种新技术--脉冲控制扩增--基于核酸扩增实现快速(20分钟)、便携、灵敏和特异的现场分子诊断。我们开发的设备重量轻，由电池供电，我们证明了我们的测试可以在资源有限的条件下进行，无需提取核酸，即使在穿戴完整的个人防护装备时也是如此。因此，分子诊断可以在非实验室环境中现场进行，而不需要提取核酸。我们预计，这项新技术有可能成为一线和现场应用的核酸检测的黄金标准方法。
Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests. Rapid and reliable on-site diagnostics are an essential part of infectious disease outbreak response and the fight against neglected tropical diseases. In this respect, molecular diagnostics represents the current gold standard. However, the vast majority of current molecular diagnostic methods (such as Polymerase Chain Reaction) are lab-bound techniques, require expensive and heavy instrumentation and are time consuming to perform. Moreover, in resource limited countries with little or no infrastructure, samples often have to be transported over long distances to the few reference laboratories capable of such diagnostics. This significantly slows down the entire diagnostic process, thus considerably delaying important decisions necessary to contain outbreaks and to initiate medical countermeasures. Until now, there are only few rapid on-site diagnostic tests for a limited number of infectious agents available and they often lack sensitivity and/or specificity. Here we present a new technology–pulse-controlled amplification–which enables rapid (<20minutes) and portable, yet sensitive and specific on-site molecular diagnostics based on nucleic acid amplification. The device we developed is lightweight, battery operated and we demonstrate that our tests can be performed under resource limited conditions and without nucleic acid extraction, even when wearing full personal protective equipment. Thus, molecular diagnostics can be carried out on-site in a non-laboratory environment without the need for nucleic acid extraction. We envision that this new technology has the potential to become a gold standard method in nucleic acid detection for front-line and in-field applications.
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影响因子：
3.8
作者：
Qu S;Shi Q;Zhou L;Guo Z;Zhou D;Zhai J;Yang R
通讯作者：
Yang R
影响因子：
2.4
作者：
Zasada, A. A.;Forminska, K.;Grunow, R.
通讯作者：
Grunow, R.
影响因子：
3.7
作者：
Poritz MA;Blaschke AJ;Byington CL;Allen L;Nilsson K;Jones DE;Thatcher SA;Robbins T;Lingenfelter B;Amiott E;Herbener A;Daly J;Dobrowolski SF;Teng DH;Ririe KM
通讯作者：
Ririe KM
影响因子：
9.8
作者：
Zou Y;Mason MG;Wang Y;Wee E;Turni C;Blackall PJ;Trau M;Botella JR
通讯作者：
Botella JR
影响因子：
3.3
作者：
Riehm, Julia M.;Rahalison, Lila;Tomaso, Herbert
通讯作者：
Tomaso, Herbert