Pulse-Controlled Amplification–A new powerful tool for on-site diagnostics under resource limited conditions

Katharina Müller, Sarah Daßen, Scott Holowachuk, Katrin Zwirglmaier, Joachim Stehr, Federico Buersgens, Lars Ullerich and Kilian Stoecker

PLOS Neglected Tropical Diseases, 2021, vol. 15, issue 1, 1-17

Abstract: Background: 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. Methods/Principle findings: 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. Conclusions/Significance: 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. Author summary: 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 (

Date: 2021
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pntd00:0009114

DOI: 10.1371/journal.pntd.0009114

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