Abstract
Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.
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Acknowledgments
This work was supported by the California Citrus Nursery Board (CCNB) project “Implementation and streamlining of the newly developed high throughput diagnostic system for citrus nurseries registration” and by the Citrus Research Board (CRB) project “Citrus Clonal Protection Program” (6100) awarded to G. Vidalakis. Additional support was provided by the US Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA), Hatch (project 1020106), and the National Clean Plant Network (NCPN) which operates under the auspices of USDA Animal and Plant Health Inspection Service (APHIS) (12-8100-1544-CA; 14-, 15-, 16- 8130-0419-CA; AP17PPQS&T00C118; AP18PPQS&T00C107). The findings and conclusions in this publication are those of the author(s) and should not be construed to represent any official USDA or US Government determination or policy. URLs to sponsors’ websites listed below.
1. CRB: https://www.citrusresearch.org
2. CCNB: http://www.ccnb.info/
3. USDA NIFA: https://nifa.usda.gov
4. NCPN: http://nationalcleanplantnetwork.org.
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Osman, F., Vidalakis, G. (2022). Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G. (eds) Viroids. Methods in Molecular Biology, vol 2316. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1464-8_16
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DOI: https://doi.org/10.1007/978-1-0716-1464-8_16
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