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Title: COMPARISON OF DNA AMPLIFICATION METHODS FOR IMPROVED DETECTION OF CANDIDATUS LIBERIBACTER SPECIES ASSOCIATED WTIH CITRUS HUANGLONGBING

Author
item LI, W - NATIONAL PLANT GERMPLASM
item TWIEG, E - NATIONAL PLANT GERMPLASM
item Hartung, John
item LEVY, L - NATIONAL PLANT GERMPLASM

Submitted to: National American Phytopathology Meetings
Publication Type: Abstract Only
Publication Acceptance Date: 4/15/2006
Publication Date: 6/1/2006
Citation: Li, W., Twieg, E.N., Hartung, J.S., Levy, L. 2006. Comparison of dna amplification methods for improved detection of candidatus liberibacter species associated wtih citrus huanglongbing. National American Phytopathology Meetings. 96(6):S67

Interpretive Summary:

Technical Abstract: Citrus huanglonbing (HLB), one of the most serious diseases of citrus worldwide, has Asian, African and American forms caused by Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus, respectively. The presumably low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to consistently detect. Although molecular diagnoses based on conventional polymerase chain reaction (PCR) and real-time PCR are usually used as confirmatory tests for symptomatic samples, the potential of PCR assays to improve detection of Liberibacter species has never been evaluated. In this study, we validated and compared four conventional PCR-based and one loop-mediated isothermal amplification (LAMP) protocol to three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays was improved compared to the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests of the presence of Liberibacters in symptomatic HLB infected samples. There were no differences of assay specificity among the methods evaluated. The TaqMan real-time PCR was at least 10 to 100 fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of Liberibacters prior to the appearance of the disease symptoms.