Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: Effect of amplicon size, melt rate, and dye translocation

Varga, Á.; James, D.

doi:10.1016/j.jviromet.2005.10.004

Cited by 94 publications

(55 citation statements)

References 33 publications

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“…The 0.1 °C stepped melt rate is known to increase the peak resolution of amplicons that melt at similar temperatures, compared with peak resolutions based on wider steps, for which the risk of ambiguity is higher (Varga & Delano 2006). Adopting the 0.1 °C stepped melt rate enabled to confirm the specificity of the primers and rule out the promotion of non-specific amplification with SYBR Green.…”

Section: Iforest -Biogeosciences and Forestrymentioning

confidence: 99%

Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

Lumia¹,

Modesti²,

Brunetti³

et al. 2018

iForest

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A high-performing detection method is essential to safeguard those countries that are still unaffected by canker stain, a devastating disease of Platanus spp. caused by Ceratocystis platani. We previously developed EvaGreen and Taqman-based Real-Time PCR to detect this pathogen, but in-depth validation is needed to guarantee users about its effectiveness and promote its utilization. In this work we present a validation study designed according to EPPO standards, focusing on the analytical and diagnostic sensitivity and specificity. We extend its technical application using SYBR Green. By performing standard curves and eight-replication-based experiments, we established the detection limit at 3 fg C. platani gDNA per PCR reaction. The repeatability and the operator-based reproducibility of the Real-Time PCR was demonstrated. Different gDNA extraction events by different operators and different gDNA extraction modalities did not affect the detection limit. The detection limit threshold cycle was earliest with SYBR Green, followed by Taqman, and EvaGreen. Spiking 6 µl DNA extractions of uninfected, necrotized wood with 3 fg C. platani gDNA confirmed the detection limit: 3 fg C. platani gDNA per PCR reaction, i.e., 0.5 fg gDNA per µl of wood extract. The assays tolerated 6 µl of necrotic C. platani-infected wood extracts without inhibition except for long-dead wood samples, while the 2 µl dose consistently allowed for successful detection. Detection of the pathogen in infected samples showed the highest diagnostic sensitivity with the SYBR Green assay. Agarose gel electrophoresis and staining was validated for visualizing amplicons, even at the detection limit. The specificity of the method was tested against 23 isolates representing the diversity of Ceratocystidaceae, and most species were not detected at 5 ng gDNA. However, some South American strains of the C. fimbriata complex were detected at doses as low as 5 fg. The method remains specific for C. platani detection as no other Ceratocystidaceae are known to colonize plane tree and the species within the geographic range of canker stain of plane tree were only detected at 500 pg or more gDNA. This work paves the way for a performance study of inter-laboratory comparisons.

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Section: Iforest -Biogeosciences and Forestrymentioning

confidence: 99%

Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

Lumia¹,

Modesti²,

Brunetti³

et al. 2018

iForest

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show abstract

“…MCA has been an effective and economical way for identification of virus stains (68), genes (69), bacterial strains (70), insect species (77), temperature validation of PCR cyclers (72), detection of translocations in lymphomas (73) and RNA interference/gene silencing (60). Thus, the presence of double peaks during MCA, is not always indicative of nonspecific amplification, and other methods such as agarose gel electrophoresis, and use of melt curve prediction software (60,67) an amplicon.…”

Section: Melting Curve Analysis (Mca)mentioning

confidence: 99%

Role of Melt Curve Analysis in Interpretation of Nutrigenomics’ MicroRNA Expression Data

Ahmed

Gouda

Hussein

et al. 2017

CGP

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“…on May 11, 2018 by guest http://aem.asm.org/ been shown to translocate between amplicons during melt curve analysis to higher-T m products (2,4,20), suggesting instability of this dye during HRM analysis. A previous T m study comparing the two chemistries demonstrated that SYBR green I melt profiles were more greatly influenced by dye, MgCl 2 , and starting DNA concentrations than melt profiles with its counterpart (12).…”

Section: Vol 73 2007 High-resolution Melt Analysis Of C Jejuni Crimentioning

confidence: 99%

High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni

Price

Smith

Huygens

et al. 2007

Appl Environ Microbiol

103

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A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.

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Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: Effect of amplicon size, melt rate, and dye translocation

Cited by 94 publications

References 33 publications

Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

Role of Melt Curve Analysis in Interpretation of Nutrigenomics’ MicroRNA Expression Data

High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni

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