Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

Myrick, Joseph T.; Pryor, Robert J.; Palais, Robert; Ison, Sean J; Sanford, Lindsay N.; Dwight, Zachary; Huuskonen, Jarkko; Sundberg, Scott O.; Wittwer, Carl T.

doi:10.1373/clinchem.2018.296608

Cited by 21 publications

(9 citation statements)

References 22 publications

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“…еqui [22]. In this regard, the following genetic markers were used for the selection of primers: sodA (gene encoding superoxide dismutase) -to differentiate S. equi from other streptococci species [23]; ICESe2 region -for differentiation of S. equi subsp. equi [24]; CRISPR-associated genes -for differentiation of S. equi subsp.…”

Section: Resultsmentioning

confidence: 99%

Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

Berdimuratova¹,

Makhamed²,

Shevtsov³

2020

Eurasian Journal of Applied Biotechnology

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As a result of the work performed, the conditions for setting up multiplex PCR with electrophoretic detection for the diagnosis of horse strangles were determined, allowing the identification and differentiation of S. equi subsp. equi in one reaction. It was found that the developed PCR protocol for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection in a “multiplex” format has a high specificity and does not lead to amplification of PCR products with DNA of closely related microorganisms, saprophytic microflora, and bacterial pathogens. The sensitivity of the protocols for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection was assessed. Diluted DNA samples of two S. equi subspecies were used as objects of research: S. equi subsp. equi and S. equi subsp. zooepidemicus. DNA samples were diluted by two-fold dilutions, starting from a concentration of 5 ng (which corresponds to 2 million 170 thousand copies in the genomic equivalent) to 1.19 * 10-6 ng (which corresponds to 0.71 copies in the genomic equivalent). DNA detection limit for S. equi subsp. equi was 66 copies in genomic equivalent or 152 fg, DNA of S. equi subsp. zooepidemicus – 132 copies in genomic equivalent or 305 fg.

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Section: Resultsmentioning

confidence: 99%

Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

Berdimuratova¹,

Makhamed²,

Shevtsov³

2020

Eurasian Journal of Applied Biotechnology

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“…Finally, by increasing concentrations of primers and rapid polymerases and using extreme temperature cycles of 0.5–2 s, robust PCR amplifications were obtained in 15–60 s without sacrificing sensitivity, specificity, efficiency, or yield. Such extreme speeds, originally obtained with bulk sample transfer between water baths [6], have now been replicated on at least three different microfluidic platforms [1,7,8].…”

Section: Introductionmentioning

confidence: 93%

“…There is a mismatch between common instrumentation and the kinetic potential of PCR. Quantitative PCR and melting analysis can be performed in less than a minute [1], but most commercial protocols recommend over an hour. PCR speeds have increased over the years, although most instruments today remain at slower, legacy speeds.…”

Section: Introductionmentioning

confidence: 99%

The kinetic requirements of extreme qPCR

Millington

Houskeeper

Quackenbush

et al. 2019

Biomolecular Detection and Quantification

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The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq ™ ), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom “extreme” PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200–500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10–60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.

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“…Recently, an extremely fast PCR with a 0.4 s·cycle −1 has been demonstrated and resulted in an incredible total reaction time of less than 15 s [ 41 ]. This fast PCR could be combined with a fast melting curve analysis to achieve a total detection time of less than 1 min [ 42 ].…”

Section: Pcr and Its Technologymentioning

confidence: 99%

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond

Zhu

Zhang

et al. 2020

TrAC Trends in Analytical Chemistry

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Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

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Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

Cited by 21 publications

References 22 publications

Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

The kinetic requirements of extreme qPCR

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond

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