Guiding our PCR experiments

Perkel, Jeffrey M.

doi:10.2144/000114283

Cited by 9 publications

(4 citation statements)

References 2 publications

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“…This method is used to directly quantify and clonally amplify the nucleic acid strands. The key difference between dPCR and traditional PCR lies in the method of measuring the quantities of nucleic acids 59 . A ‘digital’ measurement measures a certain variable quantitatively and discreetly, thanks to the fact that the sample is separated into a large number of parts and the reaction is carried out individually in each part.…”

Section: Lab‐based Methodsmentioning

confidence: 99%

“…The key difference between dPCR and traditional PCR lies in the method of measuring the quantities of nucleic acids. 59 A 'digital' measurement measures a certain variable quantitatively and discreetly, thanks to the fact that the sample is separated into a large number of parts and the reaction is carried out individually in each part. One of the limitations attributable to the dPCR is the control of the error mechanisms in the dilution phase.…”

Section: Digital Pcrmentioning

confidence: 99%

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Next‐generation methods for early disease detection in crops

Trippa,

Scalenghe,

Basso

et al. 2023

Pest Management Science

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BackgroundPlant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. Herein, in this review were presented and discussed the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations.Resultstraditional techniques of diagnosis used for plant pathogen identification are focused typically on the DNA, RNA (when molecular methods), and proteins or peptides (when serological methods) of the pathogens. Serological methods based on mainly enzyme‐linked immunosorbent assay (ELISA) are the most common method used for pathogen detection due to their high‐throughput potential and low cost. This technique is not particularly reliable and sufficiently sensitive for many pathogens detection during the asymptomatic stage of infection. For non‐cultivable pathogens in the laboratory, nucleic acid‐based technology is the best choice for consistent pathogen detection or identification. Lateral flow systems are innovative tools that allow fast and accurate results even in the field conditions, but they have sensitivity issues to be overcome. PCR assays performed on last‐generation portable thermocyclers may provide rapid detection results in situ.ConclusionThe advent of portable instruments can speed pathogen detection, reduce commercial costs, and potentially can revolutionize plant pathology. This review provides information on current methodologies and procedures for the effective detection of different plant pathogens.This article is protected by copyright. All rights reserved.

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Section: Lab‐based Methodsmentioning

confidence: 99%

Section: Digital Pcrmentioning

confidence: 99%

Next‐generation methods for early disease detection in crops

Trippa,

Scalenghe,

Basso

et al. 2023

Pest Management Science

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“…The routine detection of ctDNA is still hampered in the clinical practice not only for an intrinsic lack of sensitivity/specificity for ctDNA but also because the currently available procedures require a high level of expertise and a long execution time. 12 Moreover, the facilities necessary for the analysis, as flow cytometry and sequencing machineries, are present in specialized and certified laboratories for this kind of testing but are not always available in ordinary diagnostic laboratories. 13 Alternatively, there are many examples of sandwich hybridization protocols based on the use of highly sensitive radioactive probes 14 or gold or fluorescent nanoparticles.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Both of them show several advantages and disadvantages. , The ddPCR methodology utilizes a droplet generator that allows the partition of single pieces of DNA into droplets using an oil/water emulsion. The routine detection of ctDNA is still hampered in the clinical practice not only for an intrinsic lack of sensitivity/specificity for ctDNA but also because the currently available procedures require a high level of expertise and a long execution time . Moreover, the facilities necessary for the analysis, as flow cytometry and sequencing machineries, are present in specialized and certified laboratories for this kind of testing but are not always available in ordinary diagnostic laboratories…”

Section: Introductionmentioning

confidence: 99%

A Simple and Fast Assay Based on Carboxyfluorescein-Loaded Liposome for Quantitative DNA Detection

et al. 2020

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The development of an innovative and easy way to run assays for the quantitative detection of DNA present in biological fluids (i.e., blood, urine, and saliva) is of great interest for early diagnosis (e.g., tumors) and personalized medicine. Herein, a new quantitative assay based on the use of highly sensitive carboxyfluorescein-loaded liposomes as signal amplification systems is reported. The method has been tested for the detection of low amounts of DNA sequences. The reported proof of concept exploits a target DNA molecule as a linker between two complementary oligonucleotides. One oligonucleotide is biotinylated at its 3′ end and binds to streptavidin-coupled magnetic beads, whereas the other one is conjugated to a cholesterol molecule incorporated in the phospholipidic bilayer of the fluorescent liposomes. In the presence of the target fragment, the correct formation of a construct takes place as witnessed by a strong fluorescence signal, amplified by dissolving lipidic nanoparticles with Triton X-100. The system is able to detect specific nucleotide sequences with a very low detection threshold of target DNA (tens of picomolar). The assay allows the detection of both single- and double-stranded DNA. Studies performed in human blood serum show the correct assembling of the probe but with a reduction of limit of detection (up to ∼1 nM). This liposome signal amplification strategy could be used not only for the detection of DNA but also for other nucleic acids (mRNA; microRNA) that are difficult to be quantified by currently available protocols.

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