The kinetic requirements of extreme qPCR

Millington, A.; Houskeeper, Jessica A.; Quackenbush, John; Trauba, James M.; Wittwer, Carl T.

doi:10.1016/j.bdq.2019.100081

Cited by 26 publications

(26 citation statements)

References 24 publications

(31 reference statements)

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“…We simulated nucleic acid kinetics in high-speed melting ( 25 ) and extreme PCR thermocycling ( 26 ) experiments from previous publications. High-speed melting simulations used Equations ( 1 – 2 ) and Reaction Scheme 1.…”

Section: Methodsmentioning

confidence: 99%

Nearest-neighbour transition-state analysis for nucleic acid kinetics

Rejali

Zuiter

et al. 2021

Nucleic Acids Research

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We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl2 (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = −0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl2 compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.

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Section: Methodsmentioning

confidence: 99%

Nearest-neighbour transition-state analysis for nucleic acid kinetics

Rejali

Zuiter

et al. 2021

Nucleic Acids Research

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“…Finally, advances in the speed of amplification through techniques like "extreme PCR" will no doubt lead to a nextgeneration of rapid diagnostics [81]. Not mentioned in this review, due to scope, are systems like T2 Biosystems and Qvella, that test for bloodstream infections directly from a blood sample without the need for culture [82].…”

Section: Additional Future Directionsmentioning

confidence: 99%

Appropriate Use and Future Directions of Molecular Diagnostic Testing

Graf

Pancholi

2020

Curr Infect Dis Rep

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Purpose of Review Major technologic advances in two main areas of molecular infectious disease diagnostics have resulted in accelerated adoption or ordering, outpacing implementation, and clinical utility studies. Physicians must understand the limitations to and appropriate utilization of these technologies in order to provide cost-effective and well-informed care for their patients. Recent Findings Rapid molecular testing and, to a lesser degree, clinical metagenomics are now being routinely used in clinical practice. While these tests allow for a breadth of interrogation not possible with conventional microbiology, they pose new challenges for diagnostic and antimicrobial stewardship programs. This review will summarize the most recent literature on these two categories of technologic advances and discuss the few studies that have looked at utilization and stewardship approaches. This review also highlights the future directions for both of these technologies. Summary The appropriate utilization of rapid molecular testing and clinical metagenomics has not been well established. More studies are needed to assess their prospective impacts on patient management and antimicrobial stewardship efforts as the future state of infectious disease diagnostics will see continued expansion of these technologic advances.

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“…One heater is used for the denaturation step and the second for the reverse transcription and annealing/extension steps. The PCR tubes are then shuttled between water baths using a servo-motor operated arm controlled by a programmable microcontroller device [ 10 , 11 , 12 ]. The unit presented here can process up to 96 samples in one run and only costs approximately USD 300 to build.…”

Section: Introductionmentioning

confidence: 99%

A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings

Arumugam

Faron

et al. 2020

Diagnostics

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Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

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The kinetic requirements of extreme qPCR

Cited by 26 publications

References 24 publications

Nearest-neighbour transition-state analysis for nucleic acid kinetics

Nearest-neighbour transition-state analysis for nucleic acid kinetics

Appropriate Use and Future Directions of Molecular Diagnostic Testing

A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings

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