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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Research Project #433954

Research Project: Duplex qPCR for Simultaneous Diagnosis of HLB and Stubborn Disease

Location: Crop Diseases, Pests and Genetics Research

Project Number: 2034-22000-013-10-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Oct 1, 2017
End Date: Sep 30, 2020

1. Develop and validate a highly sensitive procedure for simultaneous detection and precise quantification of two bacterial pathogens of citrus in one sample. 2. Support additional tests needed to standardize and ensure procedure will be certified for use to detect the pathogen associated with huanglongbing (HLB) for non-regulatory surveys to monitor for presence of HLB. 3. Develop a sensitive isothermal amplification procedure for CLas using plant extract without DNA extraction.

Polymerase Chain Reaction (PCR), real-time quantitative (q) PCR, droplet digital (dd) PCR, and isothermal amplification procedures will be used and optimized employing conserved multi-copy gene sequences determined by bioinformatics from Next Generation Sequencing using MiSeq of the bacterial genomes of "Candidatus Liberibacter asiaticus" and Spiroplasma citri. Specialized software will be used to design and select primers for broad spectrum, species-specific, and population-specific identification of these pathogens. Primers along with Taqman-based probes will be incorporated for use in qPCR and ddPCR assays.