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United States Department of Agriculture

Agricultural Research Service

Research Project: Horticultural, Physiological, and Genetic Factors Affecting Sustainable Citrus Production

Location: Citrus and Other Subtropical Products Research

Title: Use of digital PCR to improve early detection of CLas infection

Author
item Mccollum, Thomas
item Levesque, Cynthia
item Godfrey, Kristine

Submitted to: Citrograph
Publication Type: Trade Journal
Publication Acceptance Date: 4/7/2016
Publication Date: 7/7/2016
Citation: McCollum, T.G., Levesque, C., Godfrey, K. 2016. Use of digital PCR to improve early detection of CLas infection. Citrograph. 7(3):48-52.

Interpretive Summary: Huanglongbing is a devastating disease of citrus caused by the bacterium Candidatus Liberibacter asiaticus. Huanglongbing has devastated the Florida citrus industry and is threatening citrus in Texas and California. Detection of Candidatus Liberibacter asiaticus infections as early as possible is key to prevention of Huanglongbing epidemics. Currently, detection of Candidatus Liberibacter asiaticus in citrus is only possible using polymerase chain reaction technology to amplify a diagnostic fragment of the pathogen nucleic acid. Real time polymerase chain reaction is the method used to identify citrus trees suspected to be infected with Liberibacter. The assay was optimized for greatest sensitivity of detection. The sensitivity of digital polymerase chain reaction for detection of Liberibacter was not greater than real time polymerase chain reaction, but is an alternative method that may have utility in confirmatory assays for Liberibacter in citrus.

Technical Abstract: Huanglongbing is a devastating disease of citrus caused by the bacterium Candidatus Liberibacter asiaticus. Huanglongbing has devastated the Florida citrus industry and is threatening citrus in Texas and California. Detection of Candidatus Liberibacter asiaticus infections as early as possible is key to prevention of Huanglongbing epidemics. Currently, detection of Candidatus Liberibacter asiaticus in citrus is only possible using polymerase chain reaction technology to amplify a diagnostic fragment of the pathogen nucleic acid. Real time polymerase chain reaction is the method used to identify citrus trees suspected to be infected with Liberibacter. However, digital polymerase reaction technology has recently become available and may provide a more sensitive alternative to real time polymerase chain reaction for Liberibacter diagnostics. In this project we determined that polymerase chain reaction primers and probes used in real time polymerase chain reaction could be used in digital polymerase chain reaction. The assay was optimized for greatest sensitivity of detection and showed a linear working range over 1 to 20,000 copies of pathogen target nucleic acid. The sensitivity of digital polymerase chain reaction for detection of Liberibacter was not greater than real time polymerase chain reaction, but is an alternative method that may have utility in confirmatory assays for Liberibacter in citrus.

Last Modified: 10/17/2017
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