Journal
PLANTS-BASEL
Volume 10, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/plants10102111
Keywords
huanglongbing; citrus; reference gene; malate dehydrogenase; qPCR; digital PCR
Categories
Funding
- Citrus Research Board [15-5050-022E]
- USDA NIFA [2015-70016-22992]
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An improved reference gene, malate dehydrogenase (MDH), has been developed for sensitive detection of the CLas bacterium in plants, suitable for diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays.
Citrus huanglongbing (HLB) disease associated with the 'Candidatus Liberibacter asiaticus' (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.
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