Droplet digital PCR for absolute quantification of plant pathogens

Authors: SELVARAJ, VIJAYANANDRAJ - Foreign Agricultural Service (FAS, USDA); MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); HAJERI, SUBHAS - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: Plant Biotechnology
Publication Type: Book / Chapter
Publication Acceptance Date: 5/30/2019
Publication Date: 11/15/2019
Citation: Selvaraj V., Maheshwari Y., Hajeri S., Yokomi R. 2019. Droplet digital PCR for absolute quantification of plant pathogens. In: Khurana S., Gaur R., editors. Plant Biotechnology: Progress in Genomic Era. Singapore: Springer. p. 583-595.
DOI: https://doi.org/10.1007/978-981-13-8499-8_25

Interpretive Summary: Precise quantitation of DNA molecules is critical for detection and diagnosis of plant pathogens. Droplet Digital Polymerase Chain Reaction (ddPCR) is the most effective PCR method due to its high sensitivity, reliability, tolerance to PCR inhibitors, and capability to obtain absolute quantitation of the target molecule without need of reference standards. Droplet Digital PCR fractionates a sample into 20,000 water-oil emulsion droplets and PCR amplification is performed on each droplet. Droplet Digital PCR uses reagents and workflows like most standard TaqMan probe-based assays. However, the massive sample partitioning and the use of Poisson statistics to determine the absolute copy number of the target molecules makes it the method of choice for disease diagnosis when the target molecule is in low concentration. Under these conditions, conventional quantitative PCR (qPCR) can often result in ambiguous results and false conclusions; whereas ddPCR will result in clear definitive conclusions. This is critical if the target is a regulated plant pathogen such as "Candidatus Liberibacter asiaticus", the bacterial agent associated with the devastating citrus disease Huanglongbing (HLB).

Technical Abstract: Droplet digital PCR (ddPCR) is a method for performing digital PCR that uses water-oil emulsion droplet technology that allows detection up to a single gene copy. Features that make it advantageous over quantitative PCR (qPCR) is ease of use, precision, specificity and reproducibility of template amplification resulting in absolute quantification. It is an ideal method for gene expression studies. ddPCR determines absolute quantification of target genes without the need of external standards, hence, it is independent of amplification efficiency bias observed with quantitative PCR. ddPCR amplification of a sample is carried out in thousands of partitioned water-oil emulsion droplets and presence or absence of the target in each droplet is determined digitally by fluorescence at the end-point of amplification. Here, ddPCR applications for plant pathogens are discussed using citrus pathogens in duplex and triplex assays.