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

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.

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

2020 ◽  
Author(s):  
Katharina Müller ◽  
Sarah Daßen ◽  
Scott Holowachuk ◽  
Katrin Zwirglmaier ◽  
Joachim Stehr ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Molecular Diagnostics ◽  
Standard Technique ◽  
Rapid Identification ◽  
Nucleic Acid Amplification ◽  
Target Sequence ◽  
Infectious Agents ◽  
Fast Heating ◽  
Heating And Cooling ◽  
Nucleic Acid Amplification Technology

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.

scholarly journals Using gel-electrophoresis and traditional PCR in RT-PCR assays for the detection of SARS-CoV-2 in resource limited areas

2020 ◽  
Author(s):  
Franz-Josef Wischmann ◽  
Frank Hünger ◽  
Uwe Cassens ◽  
Wolfgang Göhde
Keyword(s):  
Gel Electrophoresis ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Test Analysis ◽  
Resource Limited ◽  
Freeze Dried ◽  
Test Kit ◽  
Nucleic Acid Amplification Technology ◽  
Laboratory Technology

Background. qRT-PCR investigations on patient specimen seem to be the only probable laboratory technology to validate the current individual SARS-CoV-2 status correctly. This kind of nucleic acid amplification technology is available in most countries, but missing in resource limited areas with poor laboratory infrastructure. The most feasible way to build up PCR test capacities for SARS-CoV-2 in these areas is the use of conventional PCR. PCR-thermocycler and gel-electrophoresis systems could be shipped easily by parcel service to the whole world, because they are almost maintenance free and need no engineers for their installation and service. Methods. In order to reduce the complexity of sensitive reagent kits we have developed a freeze dried test kit for the traditional RT-PCR for the detection of SARS-CoV-2 in single ready to use tubes. Transport and storage of the reagents are now free of any cooling chain.Results. We have analysed 244 patient samples and compared the results to the SEEGENE Allplex™ 2019-nCoV assay. The sensitivity and specificity were 95.5% and 98.5%, respectively. Next to these performance parameters we could implement a workflow for processing 96 patient samples in parallel, handled by only 1 laboratory technician in an 8 hours working day.Conclusion. The use of conventional PCR in combination with gel-electrophoresis for test analysis is a feasible approach to make SARS-CoV-2 diagnostic available in the whole world. This is needed urgently, because SARS-CoV-2 is a question of global health and not only of the well-equipped countries.

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

2008 ◽  
Vol 57 (3) ◽  
pp. 324-331 ◽  
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.

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

2019 ◽  
Vol 116 (33) ◽  
pp. 16240-16249 ◽  
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.

Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics

2019 ◽  
Vol 141 ◽  
pp. 111448 ◽  
Author(s):  
Sang Hun Lee ◽  
Seung-min Park ◽  
Brian N. Kim ◽  
Oh Seok Kwon ◽  
Won-Yep Rho ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostics ◽  
Nucleic Acid Amplification ◽  
Next Generation

PREVALANCE OF GENETIC MUTATION ASSOSIATED WITH (RPOB) RIFAMPICIN RESISTANT AMONG THE TREATED TUBERCULOSIS PATIENTS

2020 ◽  
Vol 11 (12) ◽  
pp. 235-268
Author(s):  
Girija Vasudevan ◽  
Muthuraj M ◽  
Antony V samrot
Keyword(s):  
Drug Susceptibility ◽  
Genetic Mutation ◽  
Drug Susceptibility Testing ◽  
Nucleic Acid Amplification ◽  
Drug Resistant ◽  
Tuberculosis Patients ◽  
Resistant Tuberculosis ◽  
Resource Limited ◽  
Probe Assay ◽  
Solid Liquid

Tuberculosis (TB) is an infectious disease caused predominantly by Mycobacterium tuberculosis. India accounts for one fourth of the global TB burden. Drug-resistant tuberculosis (TB) undermines control efforts and its burden is poorly understood in resource-limited settings. Drug resistant TB is a laboratory based diagnosis and is performed either by phenotypic drug susceptibility testing using solid/liquid culture or genotypic testing for detection of resistance by Line Probe Assay/Cartridge based Nucleic Acid Amplification tests.

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