Sponsoring Institution
Agricultural Research Service/USDA
Status
ACTIVE
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Project Director
YOKOMI R K
Accession Number
433954
Project Number
2034-22000-013-010R
Dates
2017-10-01 - 2020-09-30
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
PARLIER,CA 93648
Keywords
amplification
detection
disease
greening
huanglongbing
isothermal
liberibacter
pcr
spiroplasma
Research Effort
Applied (40%)
Basic (50%)
Developmental (10%)
Classification Parameters
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|
| 212 - Pathogens and Nematodes Affecting Plants |
920 - Orange |
1160 - Pathology |
50% |
| 212 - Pathogens and Nematodes Affecting Plants |
930 - Lemon |
1170 - Epidemiology |
50% |
Goals / Objectives
- Develop and validate a highly sensitive procedure for simultaneous detection and precise quantification of two bacterial pathogens of citrus in one sample.
- 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.
- Develop a sensitive isothermal amplification procedure for CLas using plant extract without DNA extraction.
Methods (unparsed)
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.
Methods
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.