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Title: Optimizing a 16S rRNA-based double stranded DNA bindng Q-PCR method for detection of Candidatus Liberibacter species associated with plants and psyllids

Author
item Shatters, Robert - Bob
item Hunter, Wayne
item Hall, David
item McKenzie, Cindy
item Duan, Ping

Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2009
Publication Date: N/A
Citation: N/A

Interpretive Summary: Huanglongbing is the most serious citrus disease worldwide and is known to be associated with a bacterium (Candidatus Liberibacter asiaticus) that is vectored between trees by an insect known as the Asian citrus psyllid. This disease was detected in the U.S. for the first time in 2005 and threatens to end large scale commercial production of citrus. Sensitive detection methods for this bacterium are needed in citrus and psyllid samples for two reasons: 1) to monitor its movement in the U.S.; and, 2) to study the infection and disease process with the goal of using this information to develop control strategies. This paper presents a molecular detection method that is superior to currently approved methods in sensitivity and C. Liberibacter strain characterization and therefore provides an important new tool in the study and monitoring of this devastating disease.

Technical Abstract: Four Candidatus Liberibacter species (asiaticus, africanus, americanus, and psyllaurous) are phloem-limited bacteria associated with severe plant diseases. Psyllaurous is linked to psyllids yellowing in solanaceous plants while the other three species are associated with citrus greening or Huanglongbing (HLB). At least eight different PCR-based strategies have been published for detection of C. Liberibacter based primarily on 16S rRNA or the ß operon sequences. A 16S rRNA-based Taqman method is accepted as a regulatory standard in the U.S. by APHIS. This method uses species-specific primers that amplify a conserved region. Growing information on sequence variation within C. Liberibacter 16S rRNA indicates species-specific primers will miss some of the sequence variants. In this paper, we present a method with at least equal sensitivity to the Taqman assay and the ability to amplify more variant sequences and to discriminate the variations using a high resolution melt (HRM) protocol. Unique 16S rDNA primers flanking a variable region were designed to produce a 195 bp amplimer and the HRM method is demonstrated. Furthermore, a saturating double-stranded DNA dye-binding Q-PCR protocol, SensiMix HRM using EvaGreen (Trade Mark) dye, was shown to be approximately 10-fold more sensitive than SYBR Green I. High sensitivity is shown for both plant and Diaphorina citri (Asian citrus psyllids) DNA samples and detection of the much more divergent psyllaurous species was demonstrated.