Pulse-Controlled Amplification – a new powerful tool for front-line diagnostics

ABSTRACTMolecular diagnostics has become essential in the identification of many infectious 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. Here we present an 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. We could demonstrate the efficacy of this technology in two assays, one for a nosocomial context targeting MecA conferred antibiotic resistance, and one for a biothreat scenario targeting Yersinia pestis. The observed limits of detection were 10 copies per reaction (purified DNA) for MecA in a methicillin resistant Staphylococcus aureus and 434 copies per reaction (purified DNA) or 9.8 cells per reaction (crude sample) of Yersinia pestis. Thus, PCA offers a decentralization of molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed.

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

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009114 ◽

2021 ◽

Vol 15 (1) ◽

pp. e0009114

Author(s):

Katharina Müller ◽

Sarah Daßen ◽

Scott Holowachuk ◽

Katrin Zwirglmaier ◽

Joachim Stehr ◽

...

Keyword(s):

Yersinia Pestis ◽

Molecular Diagnostics ◽

Nucleic Acid Amplification ◽

Neglected Diseases ◽

Target Sequence ◽

Infectious Agents ◽

Fast Heating ◽

Resource Limited ◽

Nucleic Acid Amplification Technology ◽

Rapid Tests

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.

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A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

Scientific Reports ◽

10.1038/s41598-021-02535-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Abbas Jalili ◽

Maryam Bagheri ◽

Amir Shamloo ◽

Amir Hossein Kazemipour Ashkezari

Keyword(s):

Microfluidic Chip ◽

Point Of Care ◽

Pcr Amplification ◽

Alcohol Oxidase ◽

Nucleic Acid Amplification ◽

Adhesive Tape ◽

Microfluidic Chips ◽

Fast Heating ◽

Light Emitting ◽

Heating And Cooling

AbstractPolymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the gold nanofilm. Moreover, the gold nanofilm excited by two light-emitting diodes (LEDs) from the top and bottom sides of the chip provides fast heating of the PCR sample to 230 °C within 100 s. Such a design enables 30 thermal cycles from 60 to 95 °C within 13 min with the average heating and cooling rates of 7.37 ± 0.27 °C/s and 1.91 ± 0.03 °C/s, respectively. The experimental results demonstrate successful PCR amplification of the alcohol oxidase (AOX) gene using the rapid plasmonic photothermal cycler and exhibit the great performance of the microfluidic chip for droplet-based PCR.

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Texture Evolution Under Fast Heating and Cooling Rates of Zirconium Alloys Studied In-Situ by Synchrotron X-Ray Diffraction

SSRN Electronic Journal ◽

10.2139/ssrn.3680386 ◽

2020 ◽

Author(s):

Chi-Toan Nguyen ◽

Alistair Garner ◽

Javier Romero ◽

Antoine Ambard ◽

Michael Preuss ◽

...

Keyword(s):

Texture Evolution ◽

Zirconium Alloys ◽

X Ray Diffraction ◽

Fast Heating ◽

Cooling Rates ◽

X Ray ◽

Heating And Cooling

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Laboratory-Scale Processing and Performance Assessment of Ti–Ta High-Temperature Shape Memory Spring Actuators

Shape Memory and Superelasticity ◽

10.1007/s40830-021-00334-1 ◽

2021 ◽

Author(s):

A. Paulsen ◽

H. Dumlu ◽

D. Piorunek ◽

D. Langenkämper ◽

J. Frenzel ◽

...

Keyword(s):

High Temperature ◽

Shape Memory ◽

Detrimental Effect ◽

Wire Drawing ◽

Fast Heating ◽

Heating And Cooling ◽

Arc Melting ◽

Ω Phase ◽

And Performance

AbstractTi75Ta25 high-temperature shape memory alloys exhibit a number of features which make it difficult to use them as spring actuators. These include the high melting point of Ta (close to 3000 °C), the affinity of Ti to oxygen which leads to the formation of brittle α-case layers and the tendency to precipitate the ω-phase, which suppresses the martensitic transformation. The present work represents a case study which shows how one can overcome these issues and manufacture high quality Ti75Ta25 tensile spring actuators. The work focusses on processing (arc melting, arc welding, wire drawing, surface treatments and actuator spring geometry setting) and on cyclic actuator testing. It is shown how one can minimize the detrimental effect of ω-phase formation and ensure stable high-temperature actuation by fast heating and cooling and by intermediate rejuvenation anneals. The results are discussed on the basis of fundamental Ti–Ta metallurgy and in the light of Ni–Ti spring actuator performance.

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A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids

Journal of Medical Microbiology ◽

10.1099/jmm.0.47485-0 ◽

2008 ◽

Vol 57 (3) ◽

pp. 324-331 ◽

Cited By ~ 33

Author(s):

Alvin Stewart ◽

Benjamin Satterfield ◽

Marissa Cohen ◽

Kim O'Neill ◽

Richard Robison

Keyword(s):

Yersinia Pestis ◽

Real Time ◽

Real Time Pcr ◽

Yersinia Pseudotuberculosis ◽

Target Sequence ◽

Health Departments ◽

Pcr Assay ◽

Virulence Plasmids ◽

Taqman Probes ◽

Sporadic Disease

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

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Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904513116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16240-16249 ◽

Cited By ~ 11

Author(s):

Wei Ouyang ◽

Jongyoon Han

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Molecular Diagnostics ◽

Troponin I ◽

Direct Detection ◽

Signal To Noise Ratio ◽

Protein Detection ◽

Clinical Diagnostics ◽

Nucleic Acid Amplification ◽

Enzyme Linked Immunosorbent Assay

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

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