Poster: Biology and Disease Management: Regulatory Plant Pathology
Variation within and among laboratories in detection of Candidatus Liberibacter asiaticus using qPCR
G. MCCOLLUM (1), C. Levesque (2), M. Keremane (3), M. Kunta (4) (1) USDA-Agricultural Research Service, U.S.A.; (2) Citrus Research Board, U.S.A.; (3) USDA, ARS, U.S.A.; (4) Texas A&M University, U.S.A.
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. Diagnosis of CLas infection is based on PCR; qPCR for wide-scale surveys and conventional PCR for confirmation. 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.