|Selvaraj, Vijay Anand Raj - Foreign Agricultural Service (FAS, USDA)|
|Maheshwari, Yogita - Foreign Agricultural Service (FAS, USDA)|
|Hajeri, Subhas - Central California Tristeza Eradication Agency|
|Yokomi, Raymond - Ray|
Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Abstract Only
Publication Acceptance Date: 3/22/2016
Publication Date: 4/11/2016
Citation: Selvaraj, V., Maheshwari, Y., Hajeri, S., Yokomi, R.K. 2016. Digital PCR for detection of citrus pathogens. Conference of International Organization of Citrus Virologists. p.71.
Technical Abstract: Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. In addition, sensitivity to inhibitors in perennial plants can lead to lower quantification accuracy. Recently, droplet digital PCR (ddPCR) has been proposed as an alternative method to overcome these drawbacks. Absolute quantification by ddPCR does not rely on standards and is reported to show high tolerance to inhibitors. Therefore, the performance of ddPCR method was tested to detect and absolute quantify multiple citrus pathogens. A one-step reverse transcriptase (RT) ddPCR system optimized for TaqMan probes for Citrus tristeza virus (CTV) and Spiroplasma citri was tested. Taqman probes were designed for CTV coat protein, VT3 and T30 genotypes, and S. citri phage ORF1. Using a standardized nucleic acid sample at 200 ng/µl, ddPCR detected 6,890 copies (c) /µl coat protein; 6,690 c/µl of VT3; 6,530 c/µl of T30; and 141 c/µl of S. citri. These data showed that ddPCR directly quantified mixed pathogen samples by partitioning each of 96 wells into up to 20,000 partitions with each partition constituting an individual real-time PCR reaction. This procedure had excellent reproducibility and, thus, showed great promise to examine citrus pathogen populations in citrus trees infected with multiple pathogens or strains.