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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Citrus and Other Subtropical Products Research » Research » Publications at this Location » Publication #331433
Title: Variation within and among laboratories in detection of Candidatus Liberibacter asiaticus using qPCR

Author
item McCollum, Thomas
item LEVESQUE, CYNTHIA - Citrus Research Board
item Keremane, Manjunath
item MADHURABABU, KUNTA - Texas A&M University

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/28/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary: Huanglongbing is a disease that in the last decade has devastated the Florida citrus industry, has become widespread in Texas, and is also present in California. Huanglongbing is caused by Candidatus Liberibacter asiaticus, a bacterium that is transmitted by Asian citrus psyllids, small insects that feed on citrus phloem. Detection of Candidatus Liberibacter infections and subsequent removal of infected trees is important to reduce the risk of Huanglongbing epidemics. Currently the only way to confirm Candidatus Liberibacter infections is by polymerase chain reaction. Confirmation of Candidatus Liberibacter infections results in regulatory and management actions that have considerable economic impact. Our objective in this research was to compare results among multiple laboratories to confirm the sensitivity and diagnostic reliability of polymerase chain reaction for detection of Candidatus Liberibacter in citrus. We found very good agreement between laboratories and verified that using polymerase reaction we could detect fewer than ten bacterial cells in extract from citrus. Our results support the robustness and sensitivity of polymerase chain reaction for detection of Candidatus Liberibacter in citrus.

Technical Abstract: Huanglongbing (HLB) disease has ravaged the Florida citrus industry during the last decade and poses a serious threat to citrus production worldwide. Candidatus Liberibacter asiaticus (CLas) is the presumed causal agent of HLB in the United States. Detection and subsequent confirmation of CLas infection leads to regulatory action which has significant consequences. Diagnosis of CLas infection is based on two versions of polymerase chain reaction (PCR); qPCR for wide-scale surveys and conventional PCR for confirmation. Detection of CLas by qPCR is much maligned and the literature is replete with reports of PCR-based assays that provide greater sensitivity than does the current APHIS approved protocol. The objective of our research was to compare results among multiple laboratories using slightly different qPCR protocols to confirm the sensitivity and diagnostic reliability of qPCR. An end point dilution series of CLas-positive nucleic acid was prepared and distributed to 4 laboratories in which CLas diagnostics are routinely performed. In each laboratory qPCR diagnostics were performed using the dilution series as template, however, specific protocols typical for each laboratory varied slightly. Results were compared within and between laboratories to determine linearity, amplification efficiency, end point and consistency. Excellent agreement in results was seen among protocols within individual laboratories as well as between individual laboratories. Regression analysis indicated that regardless of protocol, PCR efficiencies were between 95 and 105%. In addition, results confirmed a Ct value of 38 for the endpoint (i.e. a single copy of target). Our results support the robustness and diagnostic reliability of the currently approved qPCR protocol.