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ARS Home » Pacific West Area » Riverside, California » National Clonal Germplasm Repository for Citrus » Research » Publications at this Location » Publication #363252
Research Project: Conservation, Management and Distribution of Citrus and Date Genetic Resources and Associated Information

Location: National Clonal Germplasm Repository for Citrus

Title: An improved reference gene for detection of “Candidatus Liberibacter asiaticus” associated with citrus huanglongbing by qPCR and digital droplet PCR assays

Author
item Keremane, Manjunath
item McCollum, Thomas
item ROOSE, MIKEAL - University Of California
item LEE, RICHARD - Retired Non ARS Employee
item RAMADUGU, CHANDRIKA - University Of California

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/2021
Publication Date: 10/8/2021
Citation: Keremane, M.L., McCollum, T.G., Roose, M.L., Lee, R.F., Ramadugu, C. 2021. An improved reference gene for detection of “Candidatus Liberibacter asiaticus” associated with citrus huanglongbing by qPCR and digital droplet PCR assays. Plants. 10(10). Article 2111. https://doi.org/10.3390/plants10102111.
DOI: https://doi.org/10.3390/plants10102111

Interpretive Summary: HLB is a serious disease of citrus. HLB is detected in regulatroy diagnostic laboratories by Real time PCR method where the assay detects both citrus DNA and the pathogen DNA simultaneously. This article reports development of a superior target gene, malate dehydrogenase (MDH), for detection of DNA from citrus and other HLB hosts to improve both sensitivity and ability to quantitate.

Technical Abstract: An improved reference gene was developed for sensitive detection of citrus huanglongbing (HLB)-associated bacteria from plants in diagnostic duplex qPCR and digital PCR assays. The mitochondrial cytochrome oxidase gene (COX) widely used as reference was found to be not suitable for dPCR assays because of high copy number. Variable numbers of mitochondrial COX observed in qPCR assays of young flush, greenhouse plants and citrus relatives suggested the need for a low copy non-variable universal reference gene for all HLB hosts. The single copy nuclear gene, malate dehydrogenase (MDH) developed here as a reference gene is amenable to data normalization. Primers and probes developed for the MDH gene fragment are conserved in most HLB hosts in the subfamily Aurantioideae. The study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.