Summary
Fluorescent-based technologies offer opportunities for developing new assays for detection, quantification, and characterization of viral isolates. According to the intrinsic characteristics of fluorescent-based tools (high specificity, sensitivity, and reliability), such type of molecular assays makes possible investigations on original studies such as evolutionary processes (including fitness measurement of isolates), quantitative epidemiology, or the analysis of synergism and antagonism between closely related isolates. The development of these tools is very simple and requires, in complement to basic molecular knowledge such as extraction, cloning, and (RT)-PCR procedures, only the identification of short specific sequence(s) in the targeted viral genome. The Single Nucleotide Polymorphism (SNP) and the ‘real-time’ RT-PCR assays are proposed as fluorescent-based tools for qualitative and quantitative viral detection, respectively. Moreover, the SNaPshot technology is described as method for isolate characterization.
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Notes
- 1.
1For samples prepared using the immunocapture procedure, sample corresponds to fixed material in ELISA plate well. The mix must include 5 μl of nuclease free water as substitute for the volume of nucleic acid extract. RT reaction occurs directly in the well of microtitration plates.
- 2.
1The simultaneous use of two enzymes is performed according to their buffer and temperature compatibility. If common buffer/temperature is not available then perform two digestion steps, and after step 2 go step 3; otherwise skip steps 3–4.
- 3.
3Includes dNTP and hot-start Taq polymerase (with 5′–3′ exonuclease activity).
- 4.
4Includes reverse transcriptase and RNase inhibitors.
- 5.
5Correspond to crude nucleic acid extracts, calibrated in vitro viral transcripts or deionized RNA-free water.
- 6.
6Includes dNTP and hot-start Taq polymerase (with 5′–3′ exonuclease activity).
- 7.
7Reaction presented for PVYN sequence detection/quantification, for PVYO application replace primers and probe by FpO, RpO, and ProbeO.
- 8.
8Includes reverse transcriptase and RNase inhibitors.
- 9.
9Includes the four labelled ddNTPs and Taq polymerase.
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Acknowledgements
The authors wish to thank Michel Tribodet, Pierre Lefeuvre, Flora Croizat, Valérie Balme-Sinibaldi, Laurent Glais and Camille Kerlan for their help during the set up of the different fluorescent-based PVY detection tools.
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Rolland, M., Delaunay, A., Jacquot, E. (2009). Fluorescent-based Techniques for Viral Detection, Quantification andCharacterisation. In: Burns, R. (eds) Plant Pathology. Methods in Molecular Biology, vol 508. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-062-1_17
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DOI: https://doi.org/10.1007/978-1-59745-062-1_17
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